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Please cite this article as: Emanuele Sasso, Seminars in Immunology, https://doi.org/10.1016/j.smim.2020.101430

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Review

New viral vectors for infectious diseases and cancer

Emanuele Sasso a,b,1, Anna Morena D’Alise a,1, Nicola Zambrano b,c, Elisa Scarselli a, Antonella Folgori d, Alfredo Nicosia b,c,* a Nouscom srl, Via di Castel Romano 100, 00128 Rome, Italy b Ceinge-Biotecnologie Avanzate S.C. A.R.L., via Gaetano Salvatore 486, 80145 Naples, Italy c Department of Molecular Medicine and Medical Biotechnology, University Federico II, Via Pansini 5, 80131 Naples, Italy d Reithera Srl, Via Castel Romano 100, 00128 Rome, Italy

A R T I C L E I N F O

Keywords: Genetic vaccine Viral vector Cancer vaccine Oncolytic virus Poxvirus Adenovirus Rhabdovirus Paramixovirus Arenavirus Herpesvirus

A B S T R A C T

Since the discovery in 1796 by Edward Jenner of vaccinia virus as a way to prevent and finally eradicate smallpox, the concept of using a virus to fight another virus has evolved into the current approaches of viral vectored genetic vaccines. In recent years, key improvements to the vaccinia virus leading to a safer version (Modified Vaccinia Ankara, MVA) and the discovery that some viruses can be used as carriers of heterologous genes encoding for pathological antigens of other infectious agents (the concept of ‘viral vectors’) has spurred a new wave of clinical research potentially providing for a solution for the long sought after vaccines against major diseases such as HIV, TB, RSV and Malaria, or emerging infectious diseases including those caused by filoviruses and coronaviruses. The unique ability of some of these viral vectors to stimulate the cellular arm of the immune response and, most importantly, T lymphocytes with cell killing activity, has also reawakened the interest toward developing therapeutic vaccines against chronic infectious diseases and cancer. To this end, existing vectors such as those based on Adenoviruses have been improved in immunogenicity and efficacy. Along the same line, new vectors that exploit viruses such as Vesicular Stomatitis Virus (VSV), Measles Virus (MV), Lymphocytic chorio-meningitis virus (LCMV), cytomegalovirus (CMV), and Herpes Simplex Virus (HSV), have emerged. Furthermore, technological progress toward modifying their genome to render some of these vectors incompetent for repli-cation has increased confidence toward their use in infant and elderly populations. Lastly, their production process being the same for every product has made viral vectored vaccines the technology of choice for rapid development of vaccines against emerging diseases and for ‘personalised’ cancer vaccines where there is an absolute need to reduce time to the patient from months to weeks or days.

Here we review the recent developments in viral vector technologies, focusing on novel vectors based on primate derived Adenoviruses and Poxviruses, Rhabdoviruses, Paramixoviruses, Arenaviruses and Herpesviruses. We describe the rationale for, immunologic mechanisms involved in, and design of viral vectored gene vaccines under development and discuss the potential utility of these novel genetic vaccine approaches in eliciting pro-tection against infectious diseases and cancer.

1. Introduction

Vaccines’ history has been a very successful one, with some vaccines even leading to disease eradication [1]. However, despite the continued success in the discovery of novel vaccines [2], development of vaccines against difficult pathogens (i.e.: HIV, Malaria, Tuberculosis) and cancer resulted in repeated failures possibly because of the lack of novel

approaches. Genetic vaccination is a novel strategy to induce an immune response

in which the gene encoding for the pathological antigen is delivered into the target cell instead of the antigen itself. While only one genetic vac-cine has been approved for use in humans [3], a large number of pre-clinical and clinical studies have been conducted with variable re-sults mainly due to the different technological platforms used. This

* Corresponding author. E-mail addresses: e.sasso@nouscom.com (E. Sasso), m.dalise@nouscom.com (A.M. D’Alise), zambrano@unina.it (N. Zambrano), e.scarselli@nouscom.com

(E. Scarselli), Antonella.Folgori@reithera.com (A. Folgori), alfredo.nicosia@unina.it (A. Nicosia). 1 Equal contribution.

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Seminars in Immunology

journal homepage: www.elsevier.com/locate/ysmim

https://doi.org/10.1016/j.smim.2020.101430 Received 29 August 2020; Received in revised form 23 October 2020; Accepted 16 November 2020

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notwithstanding, genetic vaccines have shown at least two distinct ad-vantages over more traditional approaches such as killed or inactivated microorganisms and subunit vaccines. The first one is their ability to elicit potent cellular responses, in particular cytotoxic CD8 T cells, in addition to humoral responses. This feature is mainly due to the vaccine antigen(s) being synthesized inside the target cell as a result of the expression of the vaccine encoded gene, thereby allowing efficient processing and presentation on the class I major histocompatibility (MHC-I) complex of antigen presenting cells. This feature is particularly relevant for all those diseases where antibody responses are insufficient to prevent infection and disease, or when the pathogen is highly variable in its neutralizing determinants, or in the extreme case of cancer where there is no infection to be blocked, but rather tumor cells have to be eliminated by vaccine induced cancer-specific cytolytic activity. The second advantage of genetic vaccines is their manufacturing process being always the same for every vaccine. In fact, the production process of a genetic vaccine is developed for the chemical entity (the ‘vector’) that is carrying the gene encoding for the pathological antigen, and as long as the vector remains unchanged, every novel vaccine can be manufactured with the same process.

There are three major classes of genetic vaccines: DNA vaccines, RNA vaccines and viral or bacterial vectored vaccines.

DNA vaccines are composed of a linear or a circular (plasmid) DNA molecule containing the coding sequence for the antigen of interest under the control of potent mammalian transcriptional and translational regulatory sequences. The most common form of DNA vaccines is the plasmid one which can replicate and be produced in high copy number in bacterial cells and purified to homogeneity by standard chromato-graphic methods. DNA vaccines have the advantage of easy and cost- effective production process as well as the ability to be repeatedly administered because the immune system does not react against the DNA vector. However, DNA vaccines do not efficiently transduce mammalian cells in vivo, and, therefore, their immunogenicity and ef-ficacy in large animals and humans proved to be limited. To improve cellular uptake and immunogenicity of DNA vaccines, several chemical formulations and delivery methods, including non-mechanical gene delivery, electroporation and biolistic delivery or ‘gene gun’ as well as molecular adjuvants have been developed leading to improved trans-duction and immunogenicity results [4–6].

RNA vaccines are linear RNA molecules that are transcribed in vitro, purified to homogeneity and formulated before administration. RNA vaccines contain the coding sequence of the gene of interest with suit-able translational elements for the efficient production of the encoded antigen. Like DNA vaccines they can be administered several times with no impact on their immunogenicity as no immune response against the RNA scaffold is induced. As for DNA vaccines also RNA vaccines present with the problem of low transduction efficiency in vivo, however at variance with DNA vaccines they have the advantage of skipping the steps of nuclear import, DNA transcription and nuclear export before engaging into translation. On the other end RNA molecules are intrin-sically less stable than DNA and therefore they require formulations aimed at preventing rapid degradation before and after in vivo trans-duction. As a matter of fact, recent pre-clinical data have demonstrated significant improvement in the efficiency of vaccination by using RNA molecules formulated in lipid nanoparticles which have the dual func-tion of protecting the RNA and of improving transduction in the target cells [7]. Another way to improve efficiency of RNA vaccines is to insert the RNA molecule encoding for the antigen into a viral RNA amplicon which replicates inside the target cell, thereby resulting in a higher gene dosage and antigen expression [8].

RNA vaccines are still in the early phase of clinical testing, but highly promising data have recently appeared in the literature [9], paving the way to a novel potentially highly relevant technology to be added to the armory of genetic vaccine approaches.

Viral vectored vaccines consist of viral particles whose genome has been modified to contain one or more genes encoding for the antigens of

interest. The rationale for using viruses to deliver the ‘vaccine gene’ is several fold. First of all, viruses have evolved to infect mammalian cells and express the encoded genes with high efficiency, thus solving the problem of poor in vivo transduction of nucleic acids. Most importantly, some viruses can target professional antigen presenting cells, resulting in potent priming of the immune response. Secondly, viruses (and therefore viral vectors) can grow to high titers in cell culture, thus allowing the development of large-scale production processes. Further-more, in those cases where replicating viral vectors are used, higher levels of vaccine antigens can be achieved in vivo which further boost vaccine immunogenicity. Perhaps the major limitation of some of the viral vectored genetic vaccine technologies is the presence of pre- existing immunity against the vector itself which reduces in vivo transduction and dampens the levels of immune response elicited against the vaccine antigen. Anti-vector immunity also affects the effi-ciency of repeated administrations which is impaired due to the induc-tion of neutralizing antibodies and in some cases T lymphocytes against the vector itself [10,11]. While this feature represents a major problem for the development of novel vaccines based on viral vectors, prime/-boost approaches based on two different viruses immunologically non cross-reacting (so called ‘heterologous prime/boost’) have recently shown promising results in animal models and in humans.

Because each viral species has its own biological and biochemical properties it is to be expected that different viruses will perform differently as genetic vaccine vectors. In this review we will focus on viral vectors as DNA and RNA vaccines have been reviewed elsewhere [12,13]. We will describe some of the most advanced technologies for viral vectored genetic vaccine delivery with particular emphasis on clinical data in the fields of infectious diseases and cancer. We will also describe the characteristics of these viral vector platforms including the capacity to encode large antigens, the type of immune responses induced, the possibility to re-administer them in homologous prime/-boost regimens, and their ability to be produced on a large scale. In doing so, we will attempt at providing a ‘tool box’ for interested people to explore the space of viral vectors for different applications in basic and applied vaccine research.

Since one of the advantages of viral vectors is their production pro-cess being the same for every product, we will discuss their use for the development of vaccines against emerging infectious diseases and their suitability for the establishment of ‘preparedness’ strategies.

As a major problem of viral vectors is the induction of anti-vector immunity which limits their repeated administration to boost vaccine immune responses, novel approaches of ‘heterologous prime/boost’ vaccination using serial administrations of different vectors encoding the same vaccine antigen(s) have been developed. We will review some of these approaches that were shown to improve both quantitatively, but also qualitatively the adaptive immune response, leading to higher ef-ficacy in relevant animal models, as well more potent and broad immune response in humans.

Because of their ability to induce potent cytotoxic immune responses viral vectors have recently received renewed attention as the platform of choice to develop next generation of cancer vaccines. In fact, the dis-covery of tumor-specific neoantigens to which there is no self-tolerance and the approval of effective immune Checkpoint Inhibitors (CPIs) has led to the clinical development of novel vector-based cancer vaccines. In this review we will summarise the current status of this research.

Finally, it is becoming increasingly clear that replication competent viruses do work by stimulating innate and adaptive immune response against cancer cells. Therefore, we will also discuss some of the most advanced oncolytic viruses and their merit in search for new anti-tumor therapies.

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2. Viral vectors for infectious diseases

2.1. Poxvirus vectors

Poxviruses are one of the first and most commonly used class of viral vectors for heterologous gene delivery. They are large, enveloped double-stranded DNA viruses that can accommodate incorporation of over 24,000 bp of foreign DNA sequences in their genome [14], and attenuated strains like Modified Vaccinia Ankara (MVA) were shown to efficiently and stably express recombinant antigens from an expression cassette of more than 7,5 kb [15]. This makes it possible to use them as vectors encoding for large protein antigens derived from pathogens or tumor cells. Poxvirus vectors have other favorable properties including: i) lack of persistence or genomic integration in the host due to their cytoplasmic replication; ii) low prevalence of anti-vector immunity in the global population due to the interruption of smallpox vaccination in the 1970s following its eradication; iii) acceptable safety profiles in humans, iv) proven ability to induce both humoral and cellular re-sponses without the need of an adjuvant, and v) established procedures for large production of clinical grade material. Therefore, Poxvirus vectors were used to develop several vaccine candidates against infec-tious diseases including HIV, Malaria, Tuberculosis and Influenza. The Avipoxviruses Fowlpox (FWPV) and Canarypox (CNPV), and the Orthopoxviruses Modified Vaccinia Ankara (MVA) and New York Vaccinia (NYVAC) are all artificially or naturally attenuated strains which were used as backbones to create recombinant poxvirus vaccines. FWPV, ALVAC and NYVAC have been primarily used in pre-clinical and clinical studies as HIV vaccine candidates and have been reviewed elsewhere [16], while MVA has been more extensively used as a vaccine vector against other infectious diseases. Here we will briefly describe the main features of these four vectors and some examples of the clinical results obtained with ALVAC, NYVAC and MVA.

Avipoxviruses (APVs) Fowlpox viruses (FWPV) causes disease in poultry and undergoes

abortive infection in mammalian cells (no production of progeny vi-ruses), they have a larger genome (260–309 Kb) than other known Poxviruses, that contains about 260 open reading frames.

The CNPV ALVAC is a plaque-purified clone derived from an atten-uated canarypox virus and generated by serial passages in Chicken Embryo Fibroblasts (CEF). ALVAC is significantly different from the Orthopoxvirus MVA in terms of genome size (approximately 330 Kb versus approximately 178 kbp, respectively) and the number of open readings (about 320). Many ALVAC ORFs may not function in mammalian cells, resulting in its inability to replicate in mammalian cells, and this feature underlies its improved safety profile as a vaccine vector. Both FWPV and ALVAC can be produced in clinically approved CEF. ALVAC vectored vaccines have been registered for veterinary use against several infectious diseases, including Canine distemper virus, Rabies virus, Feline leukemia virus, Equine influenza virus and West Nile virus [17].

Perhaps the most relevant clinical study with and APV vectored vaccine is the RV144 trial. This was an HIV efficacy vaccine study with more than 16,000 participants in Thailand, aimed at preventing HIV acquisition [18]. This trial involved priming the immune system with an ALVAC vector encoding HIV-1 env, gag, and pol genes (vCP1521) fol-lowed by two doses of vCP1521 plus alum-adjuvanted AIDSVAX sub-types B/E HIV envelope (env) glycoprotein (gp120). This regimen had a modest vaccine efficacy of 31 % at 3.5 years after vaccination, with a vaccine efficacy as high as 61 % during the first year (see below the Section Prime/Boost). Importantly, none of the two vaccine components showed any efficacy against HIV when used alone. The results of the rv144 trial restored a sense of optimism in the possibility to develop an efficacious HIV vaccine and raised the interest on ALVAC vectored vaccines. Subsequent in depth analyses revealed that a major component of an efficacious immune response against HIV will have to include

broad neutralizing antibodies (bNAb) and possibly antibody-dependent cell-mediated cytotoxicity (ADCC) [19].

Orthopoxviruses

NYVAC. NYVAC is an attenuated derivative of the VACV strain Copenhagen (CopV), produced by precise deletion of 18 Open Reading Frames (ORFs) from the genome of the parental virus, involved in host range, virulence and pathogenesis. NYVAC provides a high level of foreign gene expression and triggers specific immune responses to these antigens in both experimental animals and humans. NYVAC-derived vectors are able to express antigens from a broad range of species and have been used as recombinant vaccines against various pathogens and tumors. Also NYVAC has been mainly used as an HIV vaccine vector candidate, and although various protocols have been applied to induce effective immune responses, the prime/boost approach with NYVAC vectors as the boosting immunogen, has proven to be the best choice to elicit high quality antigen-specific immune responses in healthy volun-teers [16]. For example, combination of DNA and NYVAC vectors expressing the HIV Env, Gag, Pol and Nef antigens, elicited antigen-specific T cell responses in 90 % of vaccinees in contrast with the 33 % of response obtained using NYVAC alone. Vaccine-induced T cell responses in volunteers that received the DNA prime/NYVAC boost regimen were broad, polyfunctional and durable. Moreover, this regimen induced the homing of potentially protective HIV-specific CD4+

and CD8+ T cells in the gut, a major site for HIV replication and one of the major sites of CD4 T cells depletion [20,21].

MVA. Modified Vaccinia virus Ankara (MVA), was initially derived from the parental Chorioallantois Vaccinia virus Ankara (CVA) by 371 passages in primary CEF that conferred an infection phenotype with restricted host (cell) tropism [22,23], and then further cultivated beyond passage 516 and provided to the Bavarian State Institute for Vaccines to test its suitability for smallpox vaccine production [24]. The genome of MVA is 178 kb in length, with 193 open reading frames (ORFs) corresponding to 177 genes, many of which are mutated, deleted or fragmented. Mapping of these genetic alterations suggested that the inability of MVA to replicate in most mammalian cells is due to alter-ations in functions affecting host interactive proteins, including ankyrin-like genes, but also involving some structural proteins [25]. Insertion of heterologous genes at the site of deletion III in the MVA genome resulted in efficient expression and supported its general application as a safe viral vector [26]. This was confirmed by mouse immunogenicity studies with an MVA Influenza vaccine vector resulting in high levels of serum antibodies that inhibited hemagglutination by influenza A virus and cytotoxic T cell responses directed to the encoded influenza antigens. Importantly, vaccinated animals could be completely protected against a lethal respiratory tract influenza chal-lenge [27].

MVA as a poxvirus vaccine. In 2019 the FDA approved MVA, under the trade name Jynneos, to prevent both smallpox and monkeypox. The approval made MVA the first vaccine approved for monkeypox, and the first non-replicating vaccine vector approved against smallpox. Small-pox vaccines based on replicating vaccinia virus were shown to be highly effective, but also presented severe toxicity in immunocompro-mised individuals. Therefore, MVA was tested as a potentially safer smallpox vaccine. The pivotal Phase 3 study was conducted in 208 US soldiers deployed in South Korea with two doses of MVA before one dose of the established replicating-vaccinia vaccine ACAM2000, and compared that group to an ACAM2000-only control group of 213 vol-unteers. MVA vaccination did elicit a robust immune response, with a geometric mean titer of neutralizing antibodies induced by a single MVA dose (16.2) equal to that induced by ACAM2000 at 2 weeks, and 90.8 % of MVA recipients had seroconversion, compared with 91.8 % in the

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ACAM2000 group. The MVA group also produced much fewer and smaller blisters than those seen in the ACAM2000-only group [28].

MVA as a vaccine for other infectious diseases. A large number of MVA- based vaccine candidates against infectious diseases have been advanced in the clinic. One of the most studied is MVA-85A, expressing the immunodominant tuberculosis (TB) antigen 85A, and intended for use in a heterologous prime/boost strategy to prevent TB [29].

MVA-85A boosts Bacille Calmette Guerin (BCG) CD4(+) and CD8(+) T cell responses in mice, and intranasal boosting of BCG with MVA-85A afforded protection against aerosol challenge with Mycobacterium Tuberculosis (Mtb). Protection in the lung correlated with the induction of Ag 85A-specific, Interferon gamma (IFNγ) secreting T cells in lung lymph nodes [30]. Similarly, MVA-85A showed 100 % protection from Mtb challenge when used in prime/boost regimen with BCG and a Fowlpox vector encoding 85A in guinea pigs [31]. BCG/MVA-85A pri-me-boost regime also showed protective efficacy in rhesus macaques [32] and in cattle. In the latter case MVA-85A boosting resulted in protection superior to that given by BCG alone. Protection was partic-ularly evident in the lungs of vaccinated animals with bacterial loads in lymph node tissues being reduced after viral boosting of BCG-vaccinated calves compared to those in BCG-only-vaccinated animals. Analysis of vaccine-induced immunity identified memory responses measured by cultured enzyme-linked immunospot assay as well as in vitro interleukin-17 production as predictors of vaccination success, as both responses, measured before challenge, correlated positively with the degree of protection [33].

While consistent across all these animal models, efficacy of MVA-85A boost in BCG primed animals appeared to be modest, and interpretation was limited by the small number of animals tested. Nevertheless, MVA- 85A did progress into the clinic and was proven to be safe and immu-nogenic in BCG naïve as well as previously BCG vaccinated individuals. In particular, MVA-85A showed very good T cell responses (mainly CD4+) with peak value over 1000 IFN γ Spot forming cells (SFC)/million peripheral blood mononuclear cells (PBMC) in naïve individuals, and potent boost of CD4 + T cell responses in BCG experienced volunteers with up to 30 fold increased cellular immunity with respect to BCG alone (i.e.: over 2000 IFN γ SFC/million PBMC). In both cases these results are due to the high efficiency of MVA to recall primary responses due to BCG vaccination or to some pre-existing antimycobacterial immunity in BCG naïve individuals setting the ground for the use of the MVA platform technology as a booster vector rather than for the priming of an immune response in naïve individuals [29]. BCG prime-MVA85A boost immu-nisation regimen in infants was safe and induced primarily antigen-specific CD + T cells with mean peak responses of about 400 IFN γ SFC/million PBMC and a Th1 and Th17 phenotype, which is regarded as important in protection against tuberculosis [34]. Efficacy of the MVA-85A vaccine candidate was assessed in a double-blind, rando-mised, placebo-controlled phase 2b trial, in healthy infants who had previously received BCG vaccination. The vaccine was safe and immu-nogenic inducing predominantly CD4-positive T cells expressing inter-feron γ, TNFα, and interleukin 2 with some CD4-positive interleukin 17-positive T cells some of which co-expressed Th1 cytokines. Howev-er, disappointingly, the level of immune response was lower than that measured in adults (median 136 spot-forming cells per million PBMCs vs. over 2500), likely due to lower levels of primed T cells. Most importantly, the vaccine did not show efficacy as measured by reduced incidence of infection and clinical manifestations [35].

However, the remarkable ability of MVA-85A to expand primary responses in humans inspired a new wave of research in the Influenza vaccine field. In an attempt to generate a ‘Universal’ or ‘Supra-season-able’ Influenza vaccine capable of protecting against the majority of Influenza strains, T cell-based MVA vaccine vectors expressing conserved influenza antigens to be used in previously exposed in-dividuals were developed. MVA vectors expressing influenza

Nucleoprotein (NP) alone, or co-expressed with other conserved influ-enza proteins, protected mice against lethal challenge with H5N1, H7N1 or H9N2 virus. NP-expressing vectors induced high numbers of influenza-specific CD4(+) and CD8(+) T cells and high titer influenza- specific antibody responses. Higher influenza-specific CD4(+) T cell responses and NP-specific CD8(+) T cell responses were associated with increased protective efficacy [36].

An MVA vector encoding nucleoprotein and matrix protein 1 (MVA- NP + M1) was generally safe and well tolerated when delivered by intramuscular or intradermal injection in healthy volunteers [37]. Ex vivo IFNγ ELISpot T-cell responses to NP and M1 measured before vaccination had a median value of 123 SFC/million PBMC, and increased post-vaccination with peak response median 339, 443, and 1443 in low-dose intradermal, low-dose intramuscular, and high-dose intramuscular groups, respectively), with the majority of the antigen-specific T cells being CD8(+). Interestingly, similar safety and immunogenicity results were obtained in a Phase I study where the MVA-NP + M1 was manufactured on AGE1.CR.pIX Muscovy Duck immortalized cell line, paving the way toward a streamlined industrial manufacturing process [38]. Furthermore, a single intramuscular in-jection of MVA-NP + M1 at a dose of 1⋅5 × 10(8) plaque forming units (pfu) was safe and well tolerated in elderly (aged 50–85). Also in this case T cell responses were boosted significantly above baseline following vaccination with median ex vivo IFNγ ELISpot T-cell responses to NP and M1 far exceeding 1000 SFC/million PBMC. Increases were detected in both CD4(+) and CD8(+) T cell subsets. Clonality studies indicated that MVA-NP + M1 expanded pre-existing memory CD8(+) T cells, which displayed a predominant CD27(+)CD45RO(+)CD57(-)CCR7(-) pheno-type both before and after vaccination [39].

Based on these results, a phase 2a vaccination and influenza chal-lenge study was conducted in healthy adult volunteers with no measurable serum antibodies to influenza A/Wisconsin/67/2005. Two groups of eleven volunteers received either a single vaccination with MVA-NP + M1 or no vaccination. All volunteers underwent intranasal administration of influenza A/Wisconsin/67/2005 while in a quarantine unit and were monitored for symptoms of influenza disease and virus shedding. A single intramuscular injection of MVA-NP + M1 increased pre-existing T cell responses from a median value of 258–980 SFC per million PBMC. Two of 11 vaccinees and 5 of 11 control subjects devel-oped laboratory-confirmed influenza (symptoms plus virus shedding). Symptoms of influenza were less pronounced in the vaccinees and there was a significant reduction in the number of days of virus shedding in those vaccinees who developed influenza (mean, 1.09 days in controls, 0.45 days in vaccinees, P = .036). While the small numbers of volunteers in in this study did not allow to draw firm conclusions, the reduction in: a) the number of laboratory-confirmed influenza, b) the total number of symptoms recorded, and c) the virus shedding in the vaccinated group supported a protective effect of the MVA-NP + M1 vaccine against both disease severity and virus shedding [40].

A clinical trial to investigate the reduction of Influenza Like illness symptoms of a combination of MVA-NP + M1 and the seasonal vaccine was performed in volunteers aged 65 and over [41].

Most notably, Vaccinia virus was used to vaccinate over 1 billion people against smallpox, leading to the eradication of this disease in 1979 [42], hence its wide use as a carrier of genetic vaccines against several different infectious diseases and cancer. Poxvirus vectors have a long history of safe vaccination programs. However, while Poxvirus-based veterinary vaccines were successfully developed, after several clinical trials being conducted it has become clear that replica-tion incompetent Poxvirus vectors encoding heterologous antigens have lower ability to prime immune responses in humans than other viral vectors such as Adenovirus vectors [43], while showing a high capa-bility to boost both humoral and cellular responses of naturally or artificially primed immunity against a number of different pathogens. Thus, clinical research using MVA and the other Poxvirus vectors has shown a paucity of progress due to other viral vector platforms

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outcompeting them in terms of immunological potency and efficacy. However, as discussed later, the clinical experience with MVA-85A and MVA-Flu, and the encouraging results of the RV144 vaccine, prompted further consideration of poxvirus vectors, and in particular MVA, as one of the components of choice for vaccines that are based on two different vectors administered sequentially in a heterologous prime/boost regimen (see section Paragraph 3 of this Review).

2.2. Novel adenovirus vectors (non-human primate adenoviruses - NHPAd)

Adenoviruses (Ads) are small, non-enveloped viruses with an icosa-hedral nucleocapsid containing a double stranded DNA genome ranging from 26 to 45 Kb in length. Human Adenoviruses (HAds) were identified in the early 1950s as causative agents of acute upper respiratory disease. They are classified into seven species from A to G based on phylogenetic analysis of their genomic DNA sequences, pathobiology, and immuno-logical and tumorigenic properties, and each species contains multiple serotypes for a total of 57 variants. Adenoviruses have a densely packed genome with both strands being used for different transcriptional units that are transcribed in a tightly controlled temporal order. There are five early transcription units including the Early regions 1A (E1A), 1B (E1B), 2, 3, 4 (E2, E3 and E4) and intermediate/late regions which are tran-scribed at the onset of DNA replication (IX, IVa2, L4 intermediate and E2 late). The late L1-L5 mRNAs derive from a single transcription unit (major late). The majority of Adenovirus transcription units are tran-scribed by RNA polymerase II and encode for different proteins that play a role in the same step of the virus life cycle [44].

Most Adenoviruses enter cells upon binding to the Coxsackie Adenovirus receptor (CAR), but species B Adenoviruses use the ubiq-uitously expressed CD46 for cell entry [45,46]. Integrins such as αv,β3 and 5 further promote viral uptake [47]. Most of the information on Adenovirus biology comes from the studies on human Adenovirus-5 (HAdV-5) for which replication defective derivatives have been gener-ated by deletion of the E1 region to increase their safety. Further dele-tion of the E3 region was shown not to affect replication in complementing cell lines or immunogenicity, while allowing insertion of large transgene cassettes up to 7,5Kb [15]. The major determinants for Adenovirus neutralization are in the Fiber and in the hypervariable regions of the Hexon protein.

Adenoviruses infect many cell types, including quiescent cells and antigen presenting cells and their genome remains episomal conferring a safer profile compared to other viral vectors. Ad-vectored genetic vac-cines have shown in numerous systems the ability to drive robust and sustained T- and B-cell responses against the encoded transgenes, possibly due to the ability of the vector to persist in a transcriptionally active form at the site of inoculation and in lymphatic tissues [48,49]. In spite of their prolonged antigen-driven activation, Ad vector induced T-cells do not undergo exhaustion but rather maintain their function and proliferate upon booster immunization [50,51]. Very importantly, Ads can be produced on an industrial scale under good manufacturing practice (GMP) achieving titers of up to 1013 virus particles per ml (vp/mL) in continuous human cell lines adapted to grow in suspension (i.e.: HEK293, PERC6, Procell92). These features made replication-incompetent HAdV-5 one of the most preferred vectors used to date in vaccine trials.

The major challenge associated to HAdV-5 vectors that limits their clinical application is its high pre-existing adaptive immunity in humans [52–54]. Circulating anti-HAdV-5 antibodies have been shown to significantly dampen the ability of HAdV-5 vectors to transfer the gene of interest to the target tissue [55] and partially neutralize the rAd5 vaccine vector following vaccination resulting in a lower effective dose of the vaccine in Ad5 seropositive subjects. Gag-specific antibody and T lymphocyte responses were similarly blunted by baseline Ad5-specific NAbs as expected [56–58]. Also, an unexpected correlation with anti-HAd5 pre-existing immunity was described in a Phase IIb trial

(STEP trial) of a HAdV-5 vaccine expressing HIV-1 antigens where vaccination transiently increased HIV-1 acquisition rates in HAdV-5 sero-positive individuals [59].

Pre-existing immunity can be circumvented by vectors based on Adenoviruses that circulate in non-human primates (NHP) [60]. These non-human primate-derived Adenoviruses (NHPAdVs) are closely related to HAdVs both in terms of sequence homology and genome structure. The close homology in the E1 region allows trans-complementation and efficient growth of replication-deficient, E1-deleted NHPAdVs in mammalian cells already approved for use in humans that express HAdV-5 E1 such as HEK293, PerC6 and Procell92 [61].

Despite their close similarity to HAdVs, NHPAdVs are markedly different in the major neutralising determinants (hexon HVRs and fiber protein domains), resulting in a much lower seroprevalence and lower neutralizing titers in human individuals worldwide [52,62,63].

NHPAdvs were isolated from Chimpanzees (ChAd), Bonobo (PanAd), Gorillas (GAd) and Macaques (SAd), but while only two GAd vectors did reach the clinical stage (NCT04041310), none of the sAdVs progressed to the clinic. In contrast, a number of replication defective vaccine vectors were developed using ChAd and PanAd viral backbones [51, 64–66].

Similarly to Human Adenoviruses, there is a large variability in the ability to induce adaptive immune responses between different NHPAd vaccine vectors. The induced level of adaptive immunity against the encoded antigen(s) appears to correlate with the species of the parental virus, with species C being the most potent, followed by species E, while species B NHPAd, which bind to the CD46 surface molecule and more efficiently transduce dendritic cells in vitro, unexpectedly showing the lowest immunogenicity among all tested vectors [49,51,63,67]. The hierarchy of immunological potency observed between the different human and NHP derived AdVs species closely reflects the levels of an-tigen expressed in the target cell, and this in turn appears to be inversely correlated to the level of type I Interferon and other cytokines (including 10-kDa gamma interferon-induced protein, IP-10, interleukin 1 receptor antagonist, IL-1RA and interleukin 6, IL-6) induced by the different vectors in vivo [68–71]. Based on these studies, it is becoming clear that a complex balance is required between activation of the innate pathways and induction of adaptive responses, whereby an excessive induction of innate response may limit the induction of adaptive immunity against the encoded antigen by reducing the effective duration of viral vector driven transgene expression. This would imply that at variance with classical vaccines based on recombinant proteins, where adjuvants that elicit innate immunity result in increased vaccine immunogenicity, for AdVs (and maybe other viral vectored vaccines) the optimal candidates are those that have reduced capability to stimulate innate immunity pathways. Consistent with this hypothesis, species B Adenoviruses have low prevalence in both humans and chimpanzees, revealing a reduced fitness with respect to the most prevalent species C and E.

Several preclinical studies demonstrated the protective efficacy of NHPAd vectored vaccines in pre-clinical challenge mouse models of infection. More relevant data also came from experiments in larger an-imals. A single intramuscular administration of AdC68rab.gp induced sterilizing immunity against rabies infection in NHPs [72]. The AdC68rab.gp vaccine was able to induce high and sustained levels of serum neutralizing antibodies, well above the level of 0.5 IU/mL considered as a minimum adequate level of protection (WHO 2013). A similar candidate vaccine based on the ChAd155 vector is currently in Phase I clinical testing (NCT04019444).

Vaccination of calves with a PanAd vector encoding respiratory syncytial virus (RSV) antigens (PanAd3-RSV) delivered by homologous intramuscular prime/boost resulted in undetectable viral replication in the lung and no sign of pulmonary disease after a heterologous challenge with the bovine RSV (b-RSV) [73]. In this study, a single intranasal delivery of PanAd3-RSV vaccine conferred significant protection in the lung and was the only route inducing mucosal IgA and sterilizing

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protection from nasal B-RSV infection. These findings highlight the importance of identifying the relative contribution of systemic versus mucosal immune components in protection from some pathogens and may inform the development of novel and more efficacious NHPAd–based vaccination strategies tailored for different infectious diseases.

A single intramuscular administration of the species E ChAdOx1 vector encoding the spike protein of Middle East Respiratory Syndrome (MERS) Coronavirus showed induction of neutralizing antibodies, associated with almost normal clinical parameters, with no breathing irregularities or reduced lung function, limited evidence of infiltration by radiograph analysis after challenge, and no signs of gross patholog-ical lesions [74]. Vaccination resulted in no evidence of infectious virus, and only a limited presence of viral mRNA, in the lungs of vaccinated macaques. Viral mRNA and infectious virus was completely absent from animals vaccinated with a prime/boost regimen of ChAdOx1 MERS, thus confirming the superiority of a repeated intramuscular administration of NHPAd vectors as compared to a single one. Efficacy of the ChAdOx1 MERS vaccine was confirmed in camels where reduced virus shedding and nasal discharge was observed after vaccination and challenge with MERS-CoV [75].

Following the same approach, after the outbreak of the SARS-Cov2 in 2020, a ChAdOx1-vectored vaccine encoding a codon-optimised full- length spike protein of SARS-CoV-2 (ChAdOx1 nCoV-19) was con-structed and tested by vaccination and challenge in macaques. The re-sults showed that a single vaccination with ChAdOx1 nCoV-19 is effective in reducing viral loads in BAL fluid and lung tissue and in preventing damage to the lungs upon high dose challenge with SARS- CoV-2. However, despite this marked reduction in virus replication in the lungs, reduction in viral shedding from the nose was not observed. Importantly, no evidence of immune-enhanced disease was observed in vaccinated animals consistent with the skewed TH1 immune response typically induced by NHPAd [76].

Of particular relevance was the protective efficacy of a single intra-muscular injection with a ChAd3 vector encoding the Ebolavirus glycoprotein (GP), against acute lethal Zaire Ebolavirus (EBOV) chal-lenge in macaques [77]. As previously shown for other encoded anti-gens, species C ChAd3 and species E ChAd63 induced cellular immune responses were of similar quality, but of a higher magnitude in the ChAd3 vaccinated animals, consequently the protective efficacy of the ChAd63 vector was lower than that of the corresponding species C vector in this animal model, lending further support to the species C NHPAd potentially representing a superior class of vectors for vaccine use.

The first replication-incompetent NHPAd vector tested in humans was derived from the serotype ChAd63, a species E virus. Phase I studies

of ChAd63 encoding three different malaria antigens, ME-TRAP, MSP1 and AMA-1 showed excellent safety profile and low reactogenicity also at the highest intramuscular dose of 2 × 1011 vp [78,79]. ChAd63 was immunogenic at all tested doses, with T cell responses exceeding 1000 IFNγ SFC/million PBMC and antibodies against AMA-1 and MSP1 were also induced [51,78,79].

Since then, eight different NHPAd (ChAd63, ChAd3, PanAd3, ChA-dOX1, ChAd155, ChAd68 GAd20 and GAd32) have been/are being tested in clinical trials for several different indications ranging from malaria to HCV (hepatitis C virus), HIV, TB, Flu, RSV, Leishmania, Ebola, SARS-Cov2 and cancer, alone on in prime/boost regimen with MVA [80–83] (NCT04019444, NCT04041310, NCT04528641). The ChAd63 vectors encoding malaria antigens were also tested in a large number of adults’ volunteers and in neonates in malaria endemic re-gions (NCT01635647; NCT02083887), while the PanAd and ChAdOx1 vectors encoding RSV and Influenza antigens, respectively, were suc-cessfully tested in Phase 1 clinical trials in elderly populations [84,85].

With over 5000 individuals of different ages having been vaccinated in Phase 1–3 clinical trials with different NHPAd encoding various an-tigens, the NHPAd technology appears to be one of the most mature to date. The take home messages derived from the pre-clinical and clinical experience with NHPAd are as follows: i) all candidate vaccines are generally safe and well tolerated up to 2 × 1011 viral particles, ii) the vaccines induced antibodies with functional activity, iii) all vaccines induced Th1 skewed, potent T cell responses against the encoded anti-gen(s) with mean values around 103 IFNγ SFC/million PBMC by ex vivo IFNγ ELISpot (see Fig. 1), iv) the induced cellular immunity was char-acterized by elevated levels of cytotoxic CD8+ lymphocytes, v) a two- dose regimen improved antibody titers, but did not increase the level of T cell response. As the immunological hallmark of the NHPAd vector technology is its ability to rapidly induce potent CD8 + T cell responses, the use of these vectors seems to be particularly suited for vaccines against pathogens for which the development of a neutralizing antibody response remains an elusive target or for the establishment of an active immunotherapy against cancer for which the high capacity of NHPAd is instrumental to the delivery of large payloads to address the issue of tumor heterogeneity (see paragraph 5 of this Review). Furthermore, its ease of large scale manufacturing in continuous cell line cultures may prove useful for the rapid development of vaccines against emerging infectious diseases. As of today, efficacy data in humans have been produced for a Malaria vaccine based on a heterologous prime/boost with a chimp Adeno and MVA (see paragraph 3 of this Review), while clinical efficacy data with a NHPAd alone are still missing. However, ongoing trials for vaccines against RSV and SARSCov2 are likely to soon provide an answer on how broadly the NHPAd technology can be used for the development novel vaccines.

Fig. 1. T cell immunity induced in humans by different NHPAd vectors. Median T cell response measured by ex vivo IFNg ELISpot. vp: viral particles; NS: HCV Non Structural region; HIVcons: chimeric antigen composed of 14 most conserved subprotein domains of HIV-1. (Barnes et al. Science Translational Medicine, 2012 − HCV NS; O’Hara et al. J.Infect. Disease, 2012 – Ma-laria METRAP; Sheehy et al. Molecular Therapy, 2011 – Malaria MSP1; Sheehy et al. PlosOne, 2012 – Malaria AMA1; Bortwick et al. Mol. Therapy, 2014 - HIVcons; ; Ewer et al. NEJM, 2015 - Ebola GP; Folegatti et al. Lancet, 2020 - SARSCov2 Spike).

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2.3. Rhabdovirus vectors (VSV)

Rhabdoviruses (RVs) are a large family of membrane-enveloped, bullet shaped, negative sense, non-segmented, single-stranded RNA vi-ruses. They are grouped in six genera, among the most important are Lyssavirus (such as Rabies virus, RBV) and Vesiculo-virus (like Vesicular Stomatitis virus, VSV). The negative single-stranded (ss) RNA genome is about 11–16 Kb in length, and the virions range in size between 100–430 × 45–100 nm. The essential genome organization shared by all Rhab-doviruses includes five canonical genes encoding (from 3′ to 5′) the nucleoprotein (or nucleocapsid protein, N), the phosphoprotein (P), the matrix protein (M), the glycoprotein (G) and the large protein (L, RNA- dependent RNA polymerase). The viral genome is flanked by regulatory 3′ leader and 5′ trailer sequences that have terminal complementarity and contain promoter sequences to initiate replication. Each viral gene junction has conserved transcription stop and start signals separated by short untranscribed intergenic sequences [86]. The infectious ribonu-cleoprotein (RNP) complex, which is active in transcription and repli-cation, consists of the genomic RNA that is always associated with N, P and L proteins. The M protein enables the RNP complex to assemble virions at the host plasma membrane, and the transmembrane spike protein G ensures efficient virion budding, and host cell entry. The replication pathway occurs in the cytoplasm of infected cells and follows (i) cell entry, facilitated by clathrin-mediated or receptor-binding endocytosis; (ii) uncoating; (iii) transcription and translation of the viral proteins; (iv) genome replication and encapsidation; and (v) as-sembly and release (budding) [86].

Several properties of Rhabdoviruses have provided a strong rationale to explore them as a genetic vaccine vector platform. First, they can infect many species, but are rarely pathogenic in humans. Most of them are of insect or animal origin, with transmission to human populations and subsequent seroconversion uncommon, therefore they do not pre-sent pre-existing immunity in humans. Many RVs are pantropic (e.g., VSV, Maraba), entering cells through receptor-mediated endocytosis and replicating entirely within the cytoplasm with no risk for integration into the host genome and expressing their gene products to high levels. Moreover, as the family is large and diverse, different rhabdovirus strains can be employed in a heterologous ’prime/boost’ vaccine regimen, enabling repeated vector delivery.

RVs are highly immunogenic and elicit effective humoral and cellular immune responses against encoded foreign antigens while simultaneously functioning as a potent immune system adjuvant [87]. They can be engineered using reverse genetics [88] allowing the gen-eration of recombinant RV strains, which are pseudo-typed with path-ogen glycoproteins (e.g., recombinant VSV with Ebola GP [89];. Moreover, the structural and regulatory nature of the RV genome allows for transgene insertion upstream of the N gene, between genes, or downstream of the L gene. Finally, with a cloning capacity of about 6 kb, large antigens or multi-epitope open reading frames can be expressed from a single, multivalent RV recombinant virus.

Owing to its negative-sense RNA genome, rescue of recombinant virus from plasmid DNA is more challenging than rescue of DNA viruses, as it involves co-transfection of five plasmids into a permissive cell line [90]. However, reverse genetics of RVs has also enabled the engineering of attenuated RV strains that are safer than the wild type virus. This aspect has been especially crucial for neurotropic RVs such as VSV. Over the past two decades, numerous attenuation strategies have been eval-uated in preclinical model systems, including Non-human primates. These include: i) limiting viral spread by using single-cycle pseudotyped G-deleted viruses; ii) shuffling the order of the viral genes (e.g., moving the N gene from position 1–4 in the genome); preventing virus matu-ration (and thereby attenuating budding of mature virions) by trun-cating the C terminus of the G protein (e.g., from 29 to 1 amino acids); allowing for a faster host antiviral response to attenuate virus replica-tion, production and spread, by mutating the M protein (e.g., deleting amino acid 51 [91]);. Rhabdoviruses have a small size and grow to high

titer in mammalian cells that are accepted by regulatory agencies (ie.: Vero), thus enabling the virus to be produced under Good Manufacturing Practice. All these features have led to RVs development and study as genetic vaccine delivery system. However, only recently, recombinant VSV (rVSV) has come up as a valuable replication-competent vector platform.

A recombinant VSV Ebola vaccine (rVSV-EBOV) was generated using the cell culture adapted VSV Indiana strain lacking VSV-G (rVSVΔG), with the Ebola glycoprotein (EBOV-GP) placed in position 4. In the absence of the native glycoprotein, EBOV-GP acted as the vaccine an-tigen in addition to be the viral entry protein [92]. rVSV-EBOV was protective against lethal challenge with EBOV in several animal models [93]. Non-human primate (NHP) studies demonstrated 100 % protec-tion against lethal challenge with the West African EBOV-Makona strain after a single dose of rVSV-EBOV. Complete and partial protection was achieved with a single dose given as late as 7 and 3 days before chal-lenge, respectively [94].

Efficacy of the vesicular stomatitis virus-based Ebola vaccine vector in post-exposure treatment was assessed in three relevant animal models. In the guinea pig and mouse models it was possible to protect 50 % and 100 % of the animals, respectively, following treatment as late as 24 h after lethal challenge. More importantly, four out of eight rhesus macaques were protected if treated 20–30 min following an otherwise uniformly lethal infection [95]. The preclinical success of rVSV-EBOV prompted its evaluation during the recent 2014/15 outbreak. An initial placebo-controlled, double-blind phase 1/2 trial in 2015 has re-ported the rVSV-EBOV vaccine to be immunogenic. However, significant vector-associated reactogenicity has also been found, from arthritis and dermatitis to vasculitis [89,96]. In this trial, replication of the rVSV-EBOV vector was detected in synovial fluid and skin lesions, most probably due to ZEBOV-GP-specific tissue tropism. In subsequent clin-ical studies, rVSV-EBOV revealed a robust induction of neutralizing antibodies and modest CD4+ and CD8+ responses [97,98]. Most importantly, a cluster-randomized, ring vaccination trial showed that rVSV-EBOV was safe, well-tolerated, immunogenic, and, most impor-tantly, efficacious. A single IM dose of 2 × 107 plaque-forming units (PFU) was administered to individuals in direct contact with Ebola-infected patients and found to be over 90 % protective against Ebola virus infection if given as soon as possible [3]. To improve safety, second-generation rVSV-EBOV vectors were developed by employing the highly attenuated, replication competent rVSVN4CT1 backbone, and reduced virus growth was demonstrated in cell culture. The vaccine was still protective in a non-human primate Ebola virus challenge model after a single administration [99] and showed better safety in humans with the induction of an anti-Ebola neutralizing antibody response in 100 % of vaccines after two administrations [100]. However, a report of an HIV-1 trial using the rVSVN4CT1 backbone with the HIV-1 gag an-tigen coding sequence placed in position 1 of the genome was disap-pointing, with modest CD4+ levels in two-thirds of participants and low antigen-specific antibody levels in one-third of participants [101]. The development of different vaccines based on the same vector technology prompted to study the effect of pre-existing immunity against the vector. To this end, cynomolgus macaques previously vaccinated with a VSV-based Lassavirus vaccine (VSV-LASV) and challenged with LASV were vaccinated with VSV-EBOV 3 months after LASV challenge, and challenged with a lethal dose of EBOV. Despite the induction of high titers of vector-specific antibodies the animals also produced EBOV GP-specific antibodies and were completely protected from lethal EBOV challenge [102]. Thus, the presence of antibodies specific to the VSV vector do not appear to influence the protective efficacy of VSV-based vaccination, suggesting that consecutive VSV-based vaccines can be used effectively in the same population.

On December 19 2019, the FDA granted approval of the VSV-EBOV vaccine, known as Ervebo, thus making VSV the first and only viral vector that received approval as a prophylactic vaccine for use in humans. Taken together, both preclinical and clinical studies suggested

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that the major strength of the VSV vector technology lies in its attenu-ated replicative capacity and its ability to be pseudotyped with heter-ologous virus surface proteins, making this type of vectors capable of eliciting high and durable neutralizing antibody levels to surface-dis-played antigens. Furthermore, the unique advantage of the rVSV-EBOV is the rapid protection after vaccination. The use of the rVSV-EBOV vaccine in thousands of people during phase 2/3 clinical trials also showed that it is feasible to manufacture large-scale vaccine batches. These characteristics make rVSV a promising vaccine vector for emerging or outbreak-prone viral diseases. Development of second generation of more attenuated or replication incompetent version of the VSV vectors is required to broaden the spectrum of application of this technology.

2.4. Paramixovirus vectors (MEASLES virus)

The Paramixovirus family comprises major human and animal pathogens such as measles virus (MV), mumps virus (MuV), the para-influenzaviruses, Newcastle disease virus (NDV), and the highly patho-genic zoonotic hendra (HeV) and nipah (NiV) viruses. Paramyxovirus particles are pleomorphic, with a lipid envelope, nonsegmented RNA genomes of negative polarity, and densely packed glycoproteins on the virion surface.

MV is the cause of measles disease in humans, an infection of the respiratory system. Humans are the natural hosts of the virus, while no animal reservoir is known to exist.

The MV genome is non-segmented, negative-sense single-stranded RNA of approximately 16 Kb. The organization of the MeV genome comprises six genes coding for eight viral proteins arranged as 3′-N,P,V, C,M,F,H,L-5′ (with V and C overlapping P), each flanked by gene-end and gene-start sequences. Two of these proteins (V and C) are nonstructural proteins and are transcribed from the phosphoprotein gene (P). The two surface envelope glycoproteins hemagglutinin (H) and membrane fusion protein (F) are responsible for host cell binding and invasion. Three receptors for the H protein have been identified to date: complement regulatory molecule CD46, the signaling lymphocyte acti-vation molecule (SLAM) and the cell adhesion molecule Nectin-4. The RNA genome is encapsidated by the viral nucleocapsid (N) protein, resulting in the formation of a helical ribonucleoprotein (RNP) complex that serves as the template for the viral RNA-dependent RNA-polymer-ase complex composed of the viral phospho- (P) and large (L) proteins. The matrix (M) protein organizes particle assembly through interaction with both N proteins in the RNP complex and the membrane-embedded glycoprotein complexes [103]. The negative sense strand of ssRNA is used to create a positive copy, then this is used to create a new negative copy, and following multiple cycles many copies of the ssRNA are generated within the cell. The ssRNA is then mass translated by host ribosomes, producing the necessary viral proteins. The viruses are then assembled, and the cell will lyse, restarting the cycle [104].

Similar to rhabdoviruses, MV possesses an ssRNA genome of negative polarity, which has required the design of rescue systems based on reverse genetics with recombinant MV (rMV) constructs and a plasmid carrying the MV polymerase L gene transfected in a helper cell line such as HEK293 [105].

Measles vaccines are among the most effective and safest live- attenuated vaccines known and have been licensed vaccines in the US, EU, and Japan [106–109]. The live attenuated measles vaccine was developed by serial passaging of the Edmonston virus in primary renal and amnion cells, in chick embryo fibroblasts (CEFs) and in human WI-38 cells, leading to the AIK-C, Edmonston A and B, Moraten, Schwartz and Edmonston-Zagreb attenuated strains [106]. Capitalising on the high efficiency of the MV vaccine, and other MV features including: i) lack of genomic integration in the host cell due to cyto-plasmic replication, ii) large capacity for foreign gene insertion (> 6 kb), iii) stable expression of heterologous genes, the use of Schwarz, Mor-aten, or AIK-C strains for the development of live attenuated

recombinant MV (rMV) based genetic vaccines against a range of pathogens has been quite straightforward.

rMV with heterologous genes inserted between P and M or H and L were shown to express good levels of the encoded transgene and to target professional antigen-presenting cells upon intramuscular injec-tion in macaques [110].

rMV-derived vaccines have been tested in the available mouse model IFNAR− /− -CD46Ge, transgenic mice with a type I interferon receptor deficiency, but expressing the human homologue of the MV vaccine strain receptor CD46 as transgene with human tissue specificity [111] and in some cases the rMV vaccines were tested in non human primates.

rMV vaccine vectors encoding human immunodeficiency virus type 1 (HIV-1) 89.6 envelope glycoprotein, either complete or with deletion of the V3 loop induced cellular responses and neutralizing antibodies in mice and macaques. Importantly, rMV was able to raise immune re-sponses against HIV in mice and macaques with a preexisting anti-MV immunity [112]. Consistent with these findings, a candidate rMV vac-cine carrying an HIV-1 insert encoding Clade B Gag, RT and Nef (MV1-F4), induced cellular and humoral responses in mice and ma-caques, and titres of anti-F4 and rMV antibodies were boosted in vac-cinees that received a second immunization, suggesting that rMV vaccines can be readministered also in the presence of anti-vector im-munity [113].

rMV based vaccine candidates against emerging infectious diseases were also generated and tested in animals. An rMV vaccine candidate encoding envelope domains from all four Dengue virus serotypes induced neutralizing antibodies against the four serotypes of dengue virus in mice [114]. A live attenuated Schwarz MV (MVSchw-sE(WNV)) expressing the secreted form of the envelope glycoprotein from the virulent IS-98-ST1 strain of West Nile Virus (WNV) induced high levels of specific anti-WNV neutralizing antibodies in mice and protected from a lethal challenge with WNV [115].

Similarly, homologous prime/boost immunization studies in mice with rMVs expressing the spike glycoprotein of MERS-CoV in its full- length or a truncated, soluble variant induced robust levels of both rMV- and MERS-CoV-neutralizing antibodies and T cells. Vaccination with rMV MERS vectors significantly reduced viral loads in the lungs of vaccinated mice after challenge with MERS-CoV. Reduction of viral load correlated with reduced pathological alterations in the lung, indicating that rMV MERS vaccines were able to confer protection against MERS- CoV infection [116].

A recombinant live-attenuated rMV vectored vaccine against Chi-kungunya Virus was also generated. The Chikungunya virus subgenomic open reading frame encoding for structural proteins C, E3, E2, 6 K, and E1 was introduced into the Schwarz MV vaccine strain giving rise to a rMV expressing CHIKV virus-like particles comprising capsid and en-velope structural proteins (MV− CHIKV). Immunization of mice sus-ceptible to measles virus induced high titers of CHIKV antibodies that neutralized several primary isolates. Specific cellular immune responses were also elicited. A single immunization with this vaccine candidate protected all mice from a lethal CHIKV challenge, and passive transfer of immune sera conferred protection to naïve mice [117]. Homologous prime/boost vaccination with MV− CHIKV protected macaques from viremia, fever, elevated white blood cell counts, and CHIKF-associated cytokine changes upon challenge with CHIKV [118].

The MV-CHIKV and MV-F4 vaccines, which are Schwarz-strain MVs encoding CHIKV VLPs and HIV-envelope glycoprotein, respectively, have progressed into phase I clinical trials. While the HIV-vaccine trial data are not available, yet (NCT01320176), the results of the MV-CHIKV Phase I and of a randomised, placebo-controlled and active-controlled phase 2 trial have been published. These studies demonstrated the safety of the MV-CHIKV vaccine and showed induction of neutralizing antibodies even in the presence of anti-MV pre-existing immunity with a durability of up to six months [119]. In particular two administrations of 5 × 105 TCID50 MV-CHIKV induced neutralising antibodies in up to 100% of the vaccinees with no measurable rMV shedding. Moreover, the

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vaccine boost at 6 months appeared to increase neutralising antibody titres to a greater extent suggesting that a homologous prime/boost immunisation approach should be considered for further development [120].

Given the significant success of the MV vaccine, this virus was considered as a backbone for vectored vaccines against other diseases. The large database of safety data previously obtained during the cam-paigns of MV vaccination were confirmed by the Phase I and Phase II clinical results of the MV-CHIKV candidate vaccine. Very importantly, this vector achieved significant levels of neutralizing antibodies against Chikungunya virus after homologous prime/boost delivery, demon-strating not only the robustness of the administration protocol, but also the possibility to give subsequent boosts to sustain the immune response and potentially conferring lifelong protection. To date, no clinical evi-dence of the ability of the rMV technology to induce potent T cell im-munity is available and therefore its applicability to the prevention and therapy of diseases for which cellular immunity would be required re-mains to be established.

2.5. Arenavirus vectors (LCMV)

The Old World prototypic Arenavirus lymphocytic choriomeningitis virus (LCMV) is an etiological agent for human acute aseptic meningitis and grippe-like infections and is maintained in nature by lifelong persistent infections of mice. LCMV is an enveloped virus with a negative-strand RNA genome composed of two genomic RNA segments designated L and S with an approximate size of 7.2 and 3.4 Kb, respectively [121]. Each RNA segment has an ambisense coding strat-egy, encoding two proteins in opposite orientations, separated by an intergenic region. The S RNA directs synthesis of the nucleoprotein (NP) (ca 63 kDa), and the two virion glycoproteins (GP), GP-1 (40–46 kDa) and GP-2 (35 kDa), that are derived by post-translational cleavage of a precursor polypeptide, GPC (75 kDa). The L RNA segment encodes for the virus RNA-dependent RNA polymerase (L, ca. 200 kDa) and a small (11-kDa) RING finger protein (Z). Both GP-1 and GP-2, as well as NP, L, and Z, are structural proteins present in virions. The NP and viral po-lymerase are complexed with the genomic viral RNA to form ribonu-cleoprotein (RNP) complexes, which are active in virus transcription and replication. As with other negative strand RNA viruses, this RNP is the minimum unit of LCMV infectivity.

LCMV-GP mediates receptor binding and cell entry predominantly via a novel and unusual endocytotic pathway independent of clathrin, caveolin, dynamin, and actin, but dependent on alpha-dystroglycan re-ceptor (DG). LCMV-GP represents the only target for LCMV-neutralizing antibodies. The neutralizing antibody response to LCMV is extraordi-narily weak. This is an important difference between LCMV and other viral vectors and provides for an opportunity to develop Arenavirus vectors that can be re-administered in homologous prime/boost vacci-nation regimens. This peculiarity of LCMV vectors is due to an N-linked “glycan shield” on the outer globular domain of the viral envelope glycoprotein GP, which reduces antibody accessibility to critical neutralizing epitopes [122].

LCMV-induced T cell responses are broad and long-lived, and LCMV infection has been widely studied as a model of Cytotoxic T Lymphocyte (CTL)-mediated protection. LCMV also exhibits natural tropism for dendritic cells and represents the prototypic infection that elicits pro-tective CD8+ T cell immunity. These characteristics suggested that modified LCMV might serve as a vector for the induction of CTL immunity.

In preclinical models, replication incompetent recombinant LCMV (rLCMV) vaccines elicited CTL responses that were equivalent to or greater than those elicited by recombinant HAd5 or recombinant vaccinia virus in their magnitude and cytokine profiles, and they exhibited effective protection. In contrast to HAd5, rLCMV failed to elicit vector-specific antibody immunity, which facilitated re- administration of the same vector for booster vaccination. In addition,

LCMV elicited T helper type 1 CD4+ T cell responses and protective neutralizing antibodies to vaccine antigens [123].

A bivalent vaccine composed of rLCMV vectors encoding the guinea pig cytomegalovirus (GPCMV) homologs of pp65 (GP83) and gB, eli-cited potent humoral and cellular responses and was significantly more effective in reducing pup mortality than the monovalent vaccines in the GPCMV model [124].

Based on these results, an rLCMV candidate vaccine was recently tested in humans as a vaccine to prevent cytomegalovirus infection. Because LCMV vectors can accommodate transgenes of up to ~2000 bp [125,126], the Cytomegalovirus vaccine candidate (HB-101) consisted of 2 non-replicating LCMV vectors expressing the human cytomegalo-virus antigens glycoprotein B (gB) and the 65-kD phosphoprotein (pp65), respectively. In these vectors, the LCMV gene encoding the essential glycoprotein was replaced with genes encoding gB or pp65 protein and the vaccine vectors were produced in complementing HEK293 cells stably expressing the LCMV glycoprotein in trans (293-GP).

HB-101 was tested in cytomegalovirus-naive, healthy adults in a randomized, double-blind, placebo-controlled, dose-escalation Phase I trial. Vaccine was given in three intramuscular administrations and induced dose-dependent gB- and pp65-specific CD8+ and CD4+ cellular responses with mean values up to about 500 IFNγ SFC/million PBMC, dominated by pp65-specific CD8 + T cells, a high fraction of which were polyfunctional. Two administrations were sufficient to elicit dose- dependent gB-binding and cytomegalovirus-neutralizing antibodies. No detectable rLCMV vector-neutralizing response was observed in 41 of 42 vaccine recipients and as a consequence, Cytomegalovirus-specific immune responses were boosted after each administration confirming preclinical evidences of structure-based low neutralizing antibody response to LCMV [127].

rLCMV vector technology is also currently in clinical testing for the prevention of Cytomegalovirus-associated disease in transplant re-cipients (NCT03629080), and for the treatment of Papillomavirus associated cancer (NCT04180215).

Importantly, unlike replication-deficient vectors, attenuated repli-cation competent LCMV (artLCMV) targets not only dendritic, but also lymphoid tissue stromal cells expressing the alarmin interleukin-33. By triggering interleukin-33 signals, LCMV elicits effector CTL responses of higher magnitude and functionality than those induced by replication- deficient vectors [125]. The activation of the alarmin pathway may represent a discriminating feature of artLCMV especially in view of its use for active cancer immunotherapy.

While still less advanced than other genetic vaccine vectors such as MVA, VSV or NHPAd, the Arenavirus vector LCMV was shown to be safe in humans and to induce cellular (both CD4+ and CD8 + T cells) as well as neutralizing antibodies against the target pathogen. Together with this ability to induce a balanced humoral vs. cellular immune response, one of the most distinguishing features of the LCMV technology is the ability to escape vector-specific neutralizing antibodies, thereby allow-ing for repeated administrations that can boost/mature the immune response against the encoded antigen(s). Furthermore, with more than 30 different species, the Arenavirus vector technology may allow for the development of alternative vaccine backbones with more potent immunological potential and for the development of heterologous prime/boost regimens.

2.6. Herpesvirus vectors (HCMV)

Cytomegaloviruses (CMVs) are ancient β-herpesviruses that suc-cessfully coevolved with their mammalian hosts in a host-parasite relationship. Primate CMVs comprise a family of closely related, but species-specific viruses.

Human cytomegalovirus (HCMV) establishes life-long, usually asymptomatic, infection in healthy individuals, but can be life- threatening for the immunocompromised, such as HIV-infected

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persons, organ transplant recipients, or newborn infants. HCMV has broad cell tropism, including parenchymal cells and

connective tissue cells of virtually any organ and various hematopoietic cell types. Infection of epithelial cells (particularly salivary and mam-mary glands) presumably contributes to inter-host transmission. Infec-tion of endothelial cells and hematopoietic cells facilitates systemic spread within the host. Infection of ubiquitous cell types such as fibro-blasts and smooth muscle cells provides the platform for efficient pro-liferation of the virus.

Human cytomegalovirus (HCMV) has a large (236 kbp) DNA genome with more than 200 open reading frames. HCMV encodes at least 25 membrane glycoproteins that are found in the viral envelope. An HCMV pentamer complex (PC) is composed of three proteins, UL128, UL130, and UL131, that complex with gH–gL. This PC is important for infection of epithelial, endothelial cells and monocytes/macrophages. HCMV utilizes multiple host cell receptors (EGFR, PDGFR-α, CD90, NRP2 and OR14I1) to bind and infect cells, which also explains its broad tropism [128,129].

HCMV infection elicits lifelong, high-frequency and mostly effector memory (TEM) biased CD4+ and CD8 + T cell responses (up to about 10 % of the total T lymphocytes). In particular, the persistent populations of HCMV-specific CD8 + T cells (a phenomenon termed memory inflation) protect from acute, life-threatening disease, but do not mediate CMV eradication, transmission or prevent superinfection [130,131]. HCMV T-cell responses maintain functionality as they readily produce multiple effector molecules (i.e.: interferon-γ, tumour necrosis factor-α) upon stimulation, and control virus replication in vitro [132,133].

HCMV infection also elicits high-titer neutralizing antibodies re-sponses [134].

HCMV virus can be attenuated and re-designed through reverse ge-netic manipulation to improve the safety and to to express multiple heterologous antigens with a cloning capacity exceeding 6 kb [135, 136].

HCMV is also known to superinfect hosts with a history of prior exposure to the virus suggesting that CMV vector candidates have the potential to be re-administered in a homologous prime/boost regimen [137] and CMV vectors induce protective immunity in experimentally vaccinated animals with prior exposure to CMV [135,136,138].

The interest in HCMV based genetic vaccine was recently boosted after the publication of the results obtained by macaque vaccination and simian immunodeficiency virus (SIV) challenge with fibroblast adapted Rhesus CMV (RhCMV) vectors (strain 68-1) encoding SIV Gag, Env and a Rev-Tat-Nef fusion protein [135,136]. In these studies, and for the first time ever, more than 50 % of the vaccinated animals were protected from SIV induced disease. Furthermore, in all monkeys that controlled infection, initial evidence of SIV infection waned over time, and 18 months after infection, the initially infected, protected monkeys dis-played complete viral clearance and could no longer be differentiated from vaccinated monkeys not exposed to SIV. A second unprecedented finding was the observation that SIV-specific CD8 + T cells elicited by RhCMV 68− 1 vectors recognized peptides either in the context of MHC-II or the non-classical, highly conserved MHC-E molecule. These unconventional CD8 + T cell responses recognized approximately three times as many epitopes within a given antigen compared to CD8 + T cells elicited by other vaccine vectors [139], with many of these CD8 + T cells being able to recognize the same epitopes in the majority or all the vaccinated macaques (supertopes) and also when they are processed and presented by autologous SIV infected cells [140]. Importantly, these unconventional CD8 + T cells were only induced in animals immunized with the fibroblast adapted RhCMV strain 68-1. Passaging in fibroblasts results in loss of the so-called pentameric glycoprotein complex [141]. This complex facilitates infection of non-fibroblast cells such as endo-thelial, epithelial and myeloid cells, but limits viral secretion from fi-broblasts, hence the selection or mutants in fibroblasts in vitro. Fibroblast adaptation led to the deletion of the Rh157.5/4 genes which are the orthologues of the HCMV UL128/UL130 genes that encode

components of the viral pentameric complex required for viral entry into non-fibroblast cells [142]. Re-insertion of these genes into the 68-1 genome resulted in a recombinant virus (68-1.2) that only elicited MHC-I restricted CD8 + T cells, similar to wildtype RhCMV [140]. These data suggest that reprogramming a vector platform can lead to a completely different set of CD8 + T cells that differ in their epitope targeting and MHC restriction.

Despite these encouraging results, exploration of CMV as a novel vaccine vector is still at an early stage, and there are many questions still unanswered. For example, while induction of unconventional CD8 + T- cell response is very important for the prevention of SIV infection, this seemed not to be the case for protection against Mycobacterium tuber-culosis (Mtb). In fact, both RhCMV68-1- and RhCMV68-1.2-encoding Mtb antigens showed the same level of protection in macaques [138].

Direct use of RhCMV vectors in humans is not possible due to species- specific restriction of CMVs. In an effort to translate the RhCMV results into a novel vaccine vector technology for use in humans, four chimeric HCMV recombinants were generated from two fibroblast-adapted strains (Towne and Toledo) and tested in humans in a Phase 1 clinical study [143]. Molecular analysis of the chimeras revealed that they had truncated UL128 gene, and they failed to infect epithelial cells in cul-ture, consistent with a defect in pentameric complex formation. The vaccine was produced in fibroblast derived MRC5 cells and was administered to Cytomegalovirus-seronegative men. No infectious virus in urine or saliva samples of vaccine recipients was detected, suggesting that the Towne/Toledo chimeric vaccines do not establish systemic infection, and confirming that they were strongly attenuated. CD8+

T-cell responses were detected in 8 of 11 seroconverters, and responses were 0.03%–0.7% of total CD8+ T cells and peaked approximately 8 weeks after vaccination. However, none of the Towne/Toledo chimeras was able to elicit unconventional T cell responses indicating that loss of the pentameric complex alone by HCMV is insufficient for T cell reprogramming, but rather that other genes such as Rh189 (ortholog of the human US11), which inhibits responses to canonical, immunodo-minant epitopes may be required for unconventional T cell priming. Thus, more work needs to be done to generate live-attenuated HCMV vector backbones that recapitulate the unique immunological charac-teristics of fibroblast adapeted RhCMV-derived vaccines. Toward this end, a new attenuated strain of 68-1-derived RhCMV was generated by deletion of the Rh110 gene encoding the pp71 tegument protein (ΔRh110), allowing for suppression of lytic gene expression. The resulting ΔRh110 RhCMV/SIV vector was highly spread deficient in vivo, but still able to superinfect RhCMV+Rhesus Macaques and to induce potent effector-memory T cell responses. ΔRh110 RhCMV/SIV protected 59% of vaccinated animals against intravaginal (IVag) SIV-mac239challenge, providing control and progressive clearance of SIV infection [144].

Rhesus derived fibroblast adapted CMV-based vectors have acquired the unique characteristic of eliciting distinct types of CD8+ T-cell re-sponses through genetic engineering. These highly diverse CD8+ T-cell responses were contributed by CMV components through known and unknown mechanisms. CMV vectors elicited unconventional CD8+ T- cell responses recognised a large number of HLA-II and HLA-E restricted epitopes, and were shown in the SIV macaque model to afford complete viral clearance in about half of the infected animals, thus providing support for the use of this vector technology for preventing chronic in-fectious diseases that have the capability to escape antibody neutrali-zation and or to subvert the functional activity of the immune system. However, clinical validation of the CMV vector technology is still missing because no recombinant CMV vector encoding heterologous antigens has been tested in humans, yet, and fibroblast adapted atten-uated HCMV viruses were not able to induce the same type of HLA-II and HLA-E restricted CD8+ T-cell responses of the corresponding Rhesus vectors.

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3. Heterologous prime/boost with viral vectors

A homologous prime/boost vaccine strategy can be defined as a regimen of immunization where the same immunogen is administered sequentially with the objective of improving the level and longevity of the vaccine induced adaptive immunity. This is obtained by the second vaccine dose re-expanding the pool of memory B and T cells (anamnestic response) elicited by the first dose of the vaccine (primary response) [145]. Prime/boost vaccine regimens are often used such as in the case of live attenuated vaccines (ie:, oral polio vaccine), inactivated vaccines (ie:, hepatitis A virus vaccine), recombinant protein subunit vaccines (ie:, hepatitis B virus and papilloma virus vaccines) and polysaccharide vaccines (e.g., Haemophilus Influenzae type b vaccine). Several factors including delivery platform, route of immunization, doses, adjuvants, and interval between administrations can influence the outcome of prime/boost immunization approaches. The main limitation of the ho-mologous prime/boost vaccination approach based on viral vectors is linked to the induction of anti-vector immunity after the first immuni-zation. In fact, repeated administrations of the same vector often result in a reduced boosting of cellular immune response due to the elicitation of anti-vector immunity and, in particular, of neutralizing antibodies [146].

Over the past decade, several studies have shown that prime/boost immunizations can be given with unmatched vaccine delivery methods while using the same antigen, in a “heterologous” prime/boost format. Besides showing in many cases a superior immunogenicity with respect to the homologous prime/boost approach, heterologous prime/boost with two different vectors encoding for the same antigen(s) has provided for a solution to the anti-vector immunity induced by the first vaccina-tion while maximizing the host response to the vaccine antigen(s) [147].

Interest has grown in the combined use of different viral vectors and in how their sequence of administration can influence the magnitude and nature of the induced immune response. Multiple approaches have been evaluated in both animal models and humans, including FWPV- MVA, Ad-MVA, heterologous Ad-Ad, targeting a wide range of dis-eases from malaria, HIV-1, TB, RSV, HCV, Ebola to cancers. A consistent observation throughout all of these studies is the differential ability of certain vectors to prime or boost responses. For example, a large body of data now indicates that, in general, recombinant Adenoviruses, and in particular NHPAd, can prime T cell and B cell responses remarkably well (see Paragraph 2 of this Review and Fig. 1), whereas poxviruses (i.e.: MVA, ALVAC) are not efficient primers of the immune response, but are extremely good at boosting naturally or artificially primed adaptive immunity (see Paragraph 2 of this Review and Fig. 2). Therefore, an optimal regimen would use Adenovirus to prime and MVA to boost the

previously vaccine induced immune response. The heterologous prime/ boost immunization protocol based on Adenovirus and MVA has demonstrated to be a powerful strategy to induce potent and durable T cell responses as well as antibodies, and it is currently widely used in clinical trials of experimental viral vectored vaccines against different infectious diseases (World Health Organization International Clinical Trial registry, http: //apps.who.int/trialsearch).

In the previous paragraph we have described a successful example of clinical application of the heterologous prime/boost with recombinant protein and poxvirus vector [18]. In this paragraph we will focus on heterologous prime/boost with NHPAd and MVA as a mean to induce protection in animal or human challenge models as well as potent and sustained immune responses in the clinic with candidate vaccines against malaria, HCV, RSV and Ebola.

Proof of concept of improved protective efficacy achieved using heterologous prime/boost with NHPAd and MVA derived initially from ChAd63- and MVA-vectored malaria vaccines. ChAd63 and MVA viral vectors encoding the pre-erythrocytic Thrombospondin-Related Adhe-sion Protein (TRAP) antigen fused to a string of multiple CD4+ and CD8+

malaria epitopes (ME) have been tested in many studies using a standard prime/boost regime with a 8 week interval. In Phase I and II clinical trials, this vaccination regimen induced high numbers of antigen- specific T-cells in humans, and upon controlled human malaria infec-tion (CHMI) of malaria naïve adults, demonstrated 21% sterile efficacy and a delay to parasitaemia in a further 36% of vaccines [148]. The 8-week regimen induced a peak median ex vivo IFNγ ELISpot response of over 2400 SFC/106 PBMC. Protective efficacy positively correlated with the frequency of monofunctional CD8+ IFNγ+ T cells measured one day prior to challenge. Furthermore, a 67% reduction in malaria infection was measured in ChAd63/MVA ME-TRAP vaccinated adults in a malaria endemic region of coastal Kenya. In this setting, IFNγ-secreting T cell responses measured in an ex vivo ELISpot assay, correlated with pro-tection [149]. Different intervals between prime and boost and repeated MVA boosting The NHPAd/MVA regimen were also investigated and were shown to affect the kinetics and the longevity of the induced response [11].

Following these encouraging results, potent immunogenicity of other priming NHPAd vectors, including ChAd63, ChAd3, PanAd3 and ChA-dOx1 followed by MVA boost in humans has been demonstrated for HIV- 1, HCV, influenza, RSV and Ebola vaccines [80]. Fig. 2 illustrates the median T cell response measured by ex vivo IFNγ ELISpot assay induced in humans in different phase 1 clinical trials after priming with NHPAd and boosting with MVA viral vectors encoding different antigens from HCV, malaria, HIV and Ebola. Independently of the NHPAd used and the target pathogen, a potent T cell response was induced after priming with

Fig. 2. T cell immunity induced in humans by heterologous prime/boost with different NHPAd vectors and MVA. Median T cell response measured by ex vivo IFNγ ELISpot. Vector doses are indicated at the bottom of the figure and represents the number of viral par-ticles for the NHPAd and the number of plaque forming units for MVA. NS: HCV Non Structural region; HIV cons: chimeric antigen composed of 14 most conserved subprotein domains of HIV- 1. (Swadling et al. Science Translational Medi-cine, 2014 − HCV NS; Ewer et al. Nat Commun., 2013 – METRAP; Sheehy et al. Molecular Therapy, 2011 – Malaria MSP1; Sheehy et al. PlosOne, 2012 – Malaria AMA1; Bortwick et al. Mol. Therapy, 2014 – HIV cons; ; Ewer et al. NEJM, 2015 - Ebola GP).

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the addition of an MVA boost increasing the magnitude reaching median peak response up to 7000 SFC/106 PBMC.

To minimize the risks, these studies have been largely conducted in healthy young adults and indeed, the safety profile of NHPAd/MVA vaccine regimens has been excellent at both priming and boosting. Anyway, no safety concern emerged from studies in infants and babies as young as 1 week old, a key population for a malaria vaccine, where ChAd63 prime/MVA boost regimens successfully elicited high level of antigen specific T-cell responses and antibodies [150].

ChAd3 priming-MVA boosting vectors, both encoding the HCV non- structural proteins (NS3-5b), have been used to develop a purely T-cell based vaccine. Indeed, clinical testing demonstrated that strong and broad T-cell responses were generated and maintained to good levels as measured by ex-vivo assays, up to the end of follow-up, six months post boosting. A distinct, mixed phenotype of central memory and effector memory has been described for the vaccine-induced antigen specific T- cell [43] comparable to the phenotype described for the highly effective yellow fever vaccination [151,144]. A novel vaccination regimen involving serologically distinct NHPAds, ChAd3 and ChAd63, vector priming followed by MVA boosts, for simultaneous delivery of HCV NSmut antigen and HIV-1 conserved (HIVconsv) region immunogens was evaluated in a phase 1 trial [152]. Co-administration of the two vaccines by a prime/boost regimen induced high magnitude and broad T cell responses that were similar to those observed following immuni-zation with either vaccine alone demonstrating that serologically distinct NHPAds encoding HCV and HIV-1 immunogens can be safely co-administered without reducing the immunogenicity of either vac-cine. This provides a novel strategy for simultaneous targeting of path-ogens that affect the same populations.

When testing the PanAd3-RSV vaccine (a bonobo derived Adenovirus-vectored vaccine candidate that expresses a secreted form of the HRSV F protein together with the N and M2-1 proteins of Human Respiratory Syncitial Virus – HRSV) in calves, Taylor et al. compared intranasal (IN) versus intramuscular (IM) route in different regimens of homologous and heterologous prime/boost to evaluate safety, immu-nogenicity and protective efficacy against challenge with Bovine RSV (BRSV; [73]. From these studies it emerged that heterologous NHPAd prime/MVA boost vaccine regimens could completely prevent BRSV infection in the upper and lower respiratory tract of young seronegative calves. All vaccinated animals showed good level of serum neutralizing antibodies and T-cell responses that rapidly expanded after BRSV chal-lenge. This work provided evidence that a safe and protective RSV vaccine should be based on the ability to activate both B- and T-cells in a concerted manner. The phase 1 study showed that the vaccine was safe and well tolerated also when delivered by the IN route, and no vaccine-related serious adverse events occurred. Both intranasal and intramuscular administration of the vaccine led to increased neutral-izing antibody titers above the high levels of pre-existing anti-RSV an-tibodies derived from repeated seasonal exposure to the virus. Circulating anti-F immunoglobulin G (IgG) and IgA antibody-secreting cells (ASCs) were observed after the IM PanAd3-RSV prime and IM MVA-RSV boost. RSV-specific T-cell responses were increased after the IM PanAd3-RSV prime and were most efficiently boosted by IM delivery of MVA-RSV [153].

A ChAd3 vector expressing the Ebola Zaire glycoprotein, ChAd3- EBO-Z, went through accelerated safety and efficacy testing with un-precedented speed due to the 2014 West African Ebola virus outbreak. Rhesus macaques administered a single dose of the vaccine were pro-tected from an acute EBOV challenge 5 weeks later, and when boosted with MVA expressing EBOV glycoprotein were still protected at 10 months, while ChAd3-EBO-Z alone was only partially protective, demonstrating the value of the heterologous prime/boost in providing long term protection [77]. In humans a single vaccination with a biva-lent ChAd3-EBO glycoprotein Zaire and ChAd3-EBO glycoprotein Sudan, in a 1:1 ratio, at the higher dose of 2 × 1011vp induced high level of antigen-specific T-cells and sustained antibody responses, with no

vaccine-related serious adverse event. Boosting with MVA significantly increased the level of antigen-specific T-cells and antibody titers above the putatively protective levels [154,155]. Interestingly, as for the ChAd63/MVA encoding ME-TRAP, different intervals between priming and boosting immunizations showed to affect the kinetics and magni-tude of induced immunity, thus suggesting that the regimen should be adjusted depending on the need: a shorter interval might be useful to obtain a faster protection during an outbreak, while a longer interval might be suited in preventative mass campaigns.

Another Ebola vaccine based on priming with human adenovirus Ad26 encoding Ebola GP (Ad26.ZEBOV) and boosting with MVA (MVA- BN-Filo, based on Bavarian Nordic MVA-BN technology) was found to be safe and immunogenic in phase 1 and 2 trials in adults and children [156–159]. Phase 3 studies of the Ad26.ZEBOV/MVA-BN-Filo vaccine, produced by Johnson & Johnson are ongoing in multiple countries. This vaccine was deployed in the 2019 DRC Ebola outbreak (WHO. Ebola virus disease democratic Republic of the Congo) and has recently received marketing authorization by EMA.

As an example of a different viral vectors combination, an heterol-ogous prime/boost vaccine using rVSV and Ad5 expressing the GP of the Makona variant of the Ebola virus has been shown to be safe in healthy adult volunteers through phase 1/2 trials and has been licensed in Russia [160]. In this strategy the Ad5-EBOV serves as a boost to the VSV-EBOV prime vaccination. A post-market surveillance active phase 4 trial in Guinea and Russia with 2,000 participants has been conducted to confirm immunogenicity, epidemiological efficacy, and safety of this vaccine.

While some of the most recently developed viral vectors such as VSV, MV and LCMV can be re-administered without losing the ability to boost adaptive immunity against the encoded antigen(s), for others, like NHPAd, short term re-administration is not effective thereby limiting their use to those indications where short term protection is sufficient to prevent disease. Surprisingly, even serologically distinct Adenoviruses such as human Ad6 and NHP ChAd3 do not synergize when adminis-tered sequentially in humans, suggesting that classical anti-vector neutralizing antibodies are not the only mechanism preventing multi-ple administrations of this type of vectors [10]. Initially devised to overcome the issue of anti-vector immunity, heterologous prime/boost with NHPAd and MVA vectors has shown to induce superior immunity in terms of potency, breadth, longevity and, most importantly quality, paving the way for the development of vaccines against diseases requiring longer term immunity such as Ebola or higher quality of the cellular immunity such as cancer (as described in Paragraph 5 of this Review).

4. The Concept of preparedness

Epidemics and pandemics such as H1N1 (‘Spanish’) and H5N1 (‘Avian’) Influenza, HIV, Ebola, Zika and very recently SARS-CoV-2 have raised the awareness of global threats to human health posed by known as well as newly emerging pathogens and can provide the impetus to prepare against future pandemics by promoting the development of vaccine platforms that can tackle the challenges of outbreak situations. New platforms, such as viral vectors and nucleic acid based vaccines meet the prerequisites to provide solutions for some of these challenges by representing highly versatile technologies that allow fast vaccine manufacturing [161].

A number of challenges needs to be faced when developing a vaccine for a novel pathogen responsible for an outbreak: i) the unpredictable nature of the emerging pathogen does not allow to rely on the knowl-edge of the preferred antigen(s) and of defined immune correlates of protection; ii) reduced vaccine construction and production time as well as regulatory approval and large scale manufacturing time are of essence to limit spread of the infection; iii) the route of administration needs to be reliable and easy to perform in an outbreak situation, arguing for established routes of vaccination such as oral or intramuscular

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administration; iv) production capacity is often insufficient to support global vaccination, requiring innovative solutions and technologies that enable fast production of large amounts of vaccine.

As discussed in the previous paragraphs, most viral vectors present a number of features that provide for a solution to all of the above issues and render them a convenient platform to develop a vaccine prepared-ness strategy. First of all, viral genomes can be manipulated to express the antigen(s) of choice and the ability to stably accept relatively large insertions in their genome supports the development of a large variety of vaccines. Secondly, viral vectored vaccines are genetic vaccines, and, therefore, they deliver the target antigen(s) as genetic information potentially allowing faithful antigen generation with correct protein folding, multimerization and post-translational modifications [4,162, 163]. For the same reason, viral vectored vaccine antigens are efficiently processed and presented in the context of MHC-I and MHC-II context, thus being able to induce potent cellular immunity besides eliciting antibody responses against native and conformational epitopes. Strate-gies to achieve replication incompetency or attenuation of modern viral vectors generally ensure a good safety profile of viral vector-based vaccines. For most commonly employed viral vector-based vaccines, high yield production processes with means of upscaling have been established, supporting the use of these technologies for pandemic set-tings. Finally, and perhaps most importantly, the biological and physi-cochemical properties of a viral vector are always the same independently from the encoded antigen. Therefore, once a construction method and a production process is developed for a first vaccine, all the new ones will follow exactly the same procedure, thus significantly reducing time to first in man. For the same reason quality control and toxicology tests could in principle be reduced at a minimum, once a reasonable of different vectors have been extensively tested and shown to be safe in pre-clinical studies and in humans.

NHPAd vectored vaccines provide a good example of a suitable vaccine preparedness platform, which has been identified as one of significant interest by the WHO R&D Blueprint process [164].

Importantly, high-capacity NHPAd-vectors can be loaded with mul-tiple genes encoding for both B- and T-cell antigens, thus avoiding the step of deciphering the immune correlates by eliciting a full spectrum of immune responses. For enveloped viruses, the expected vaccine-induced immune responses will be mainly functional antibodies against the encoded viral glycoproteins; nevertheless, an added value of the NHPAd-vector platform is its clinically documented ability to efficiently elicit potent and durable antigen-specific cell mediated immune re-sponses, which contribute to help and sustain the humoral responses and to eliminate infected cell reservoirs, reducing the disease burden. NHPAd do not need adjuvants, making them suitable for a single dose intramuscular administration approach to rapidly induce protective immunity during an outbreak. Rapid construction methods based on bacterial artificial chromosomes (BAC) vectors and sofisticated recom-bineering techniques permit vector construction in less than a week [165,166]. Highly productive cell substrates have been developed (PERC6, Procell92, M9) achieving over one dose/mL with endogenous repression systems of transgene expression preventing rearrangements events during vector propagation and production. Several dozens of large batches of GMP manufactured NHPAd have been produced with no evidence of reversal of the replication incompetent phenotype, and well defined release tests have reproducibly assessed the robustness of the upstream and downstream processes potentially allowing to perform in the future only a restricted number of release tests including sterility, identity and potency, thereby significantly abating production time and cost of goods. Large batches of GMP grade cells can be produced and conveniently stored for immediate use for NHPAd seed infection in case of an emergency without the need of lengthy amplification steps.

Non clinical GLP toxicology studies after intramuscular vaccine de-livery were conducted in several animal species with different NHPAds and hAds [167–169]. Animal species was chosen depending on the type of immune response the vaccine was meant to induce (Ab or T cell) and

availability of assays to demonstrate vaccine take. Study design included repeated administration of a single vector or heterologous prime/boost regimens, often with N + 1 design, and whenever the species allowed were conducted at the target maximal human dose. All studies gave consistently similar results, with vaccines locally well tolerated and no evidence of systemic toxicity apart minor transient increase in body temperature and decrease in body weight 24 h post vaccination and minor and fully reversible inflammatory changes in hematochemical parameters were also noted. Several bio-distribution studies were also conducted in mice and rats with various NHPAds and studies showed identical findings; vector genomes (qPCR) were consistently detectable in the injected muscle and vp in draining iliac lymph node and with copy numbers decreasing over time. The transgene encoded by the different NHPAd-vectored vaccines is not expected to have an impact on the bio-distribution since it will only be expressed upon infection of target cells. Therefore, waiver from Regulatory Agencies to perform bio-distribution studies and possibly in vivo toxicology studies for the NHPAd platform when used by intramuscular route might be asked to accelerate clinical development and vaccine deployment.

Because of the many Phase I to III clinical trials being conducted in over 6000 individuals of all ages with different NHPAd, including ChAd63, ChAd3, ChAdOx1, PanAd3 and ChAd155 encoding for various antigens from Malaria, HCV, HIV, TB, Flu, RSV, Ebola and SARS-CoV-2 and the resulting safety and immunogenicity data (see Figs. 1 and 2) it would be reasonable to pre-select the dose for every vaccine further reducing time and costs of clinical development. Thus the target vacci-nation schedule for NHPAd-based genetic vaccines against emerging infectious diseases will likely consists in a single intramuscular admin-istration of 1 × 1011vp or 2 × 1011 viral particles. Based on previous clinical studies, the antibodies against the encoded viral antigens are expected to peak between 4 and 6 weeks after immunization, while antigen-specific T cell responses are expected to peak between 2 and 4 weeks after vaccination. To guarantee sustained immune responses over time, a homologous boost with the same vaccine vector can be admin-istered from 6 to 12 months after vaccination.

Room temperature formulations [170] and multi-dose vaccine for-mats can be used to help guide vaccine developers, manufacturers, distributors, and purchasers. Finally, the large body of knowledge about the NHPAd production process and cell substrate argue in favor of a model whereby large production facilities could be built according to the same structure and production process and be ready for every new vaccine without the need of continuously re-adapting the industrial investments.

The high density of population in several urban and suburban re-gions and the high mobility of people worldwide have significantly increased the risk of spreading pathogens. This risk is further increased by the climate change that influences the prevalence of pathogen- bearing vectors. The 2020 SARS-Cov-2 pandemic has raised awareness of the need to develop vaccines in months rather than years. To this end the genetic vaccine technologies offer several advantages over more traditional approaches such as live attenuated or whole inactivated microorganisms and vaccines based on recombinant proteins, including ease of construction, short turnaround time and standardized produc-tion process. Spurred by the urgent need to face the COVID19 pan-demics, many of these genetic vaccine platforms are being tested, with some of them already reaching the last step of clinical development. This acceleration will likely contribute to assess the maturity of these tech-nologies and their suitability also for other vaccines.

5. Renaissance of Cancer Vaccines and the use of viral vectors

5.1. From the discovery of the antitumoral activity of check point blockade to the significance of cancer neoantigens

Since the pioneering studies by William B. Coley [171], the prospect of eliciting tumor- specific cytotoxic T lymphocytes (CTL) for selective

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destruction of cancer cells prompted extensive efforts to develop cancer vaccines. However, while the use of vaccines for preventing infectious disease represents a story of great success, development of effective cancer vaccines revealed to be more difficult, primarily due to the immune-suppressive microenvironment established by cancer cells and by the low immunogenicity of autologous Tumor Associated Antigens. Indeed, therapeutic cancer vaccines only sporadically achieved clinical benefits, with only one product, Sipuleucel-T, being approved by the U. S. Food and Drug Administration (FDA) on April 29, 2010, to treat asymptomatic or minimally symptomatic metastatic Hormone Re-fractory Prostate Cancer [172] and many approaches having failed, including the MAGE-A3 Phase III MAGRIT study, the largest ever cancer vaccine trial in NSCLC patients [173].

However, recent discoveries have allowed to address the two major challenges for the development of effective cancer vaccines: i) the need to overcome the immunosuppressive tumor microenvironment (TME), ii) the selection of the right tumor antigens. In this paragraph we will focus on a novel generation of virus vectored cancer vaccines based on a novel category of tumor antigens, the so called tumor ‘neoantigens’.

The biggest challenge for developing an effective cancer vaccine and likely one of the main causes of the limited success of the previous cancer vaccination approaches is the immunosuppressive microenvi-ronment induced by the malignant machinery. It is now clear that, in order to exist within the context of a competent immune system, cancer cells need to establish a “favorable microenvironment” that impairs the ability of immune cells to recognize and eliminate them. One of the strategies that cancer cells adopt to curtail anti-tumor immunity is the expression of multiple inhibitory ligands engaging the so called ‘immune checkpoint’ molecules expressed on immune cells, thereby delivering inhibitory signals that block T cell activation and survival.

The identification of immune checkpoints and their ligands and the finding that they are overexpressed on tumor infiltrating T lymphocytes and on tumor cells, respectively, has led to the development of the so called ‘checkpoint inhibitors’ (checkpoint inhibitors, CPI) and their use in immune checkpoint therapy [174,175]. Immune checkpoint therapy is a novel therapeutic approach where passively transferred antibodies targeting immune checkpoints or their ligands block the inhibitory signals delivered by tumor cells and unleash the full power of anti-tumor T cells. Several antibodies targeting the immune checkpoints or their ligands (CTLA4, PD-1, and PD-L1) have shown remarkable activity and are currently approved for use in a variety of cancer indications. These therapies for the first time in the field of clinical oncology resulted in long-term remissions in advanced cancers regarded as complete cures [176–178]. Preclinical and clinical data have conclusively shown that the mechanism of tumor blockade or even clearance is the re-activation

of anti-tumor T cells spontaneously elicited by the patients’ immune system, but rendered ineffective by the immunosuppressive microenvi-ronment established by cancer cells through the CPI/CPI ligand pathway.

While single-agent anti-CTLA-4 and anti-PD-1 have shown an un-precedented rate of success in different cancer indications, its efficacy remains somewhat limited to only a fraction of treated patients (ranging from 20 % to 40 %; [179–181], thereby leaving room for improvement through novel combination therapies. Indeed, checkpoint blockade immunotherapy relies on the presence of antigen-specific T cells within tumor tissue. Patients with a high tumor mutational burden (TMB) may express more neoantigens and therefore could be more easily recognized by the immune system. In line with this hypothesis, tumors with increased TMB, such as melanoma and non-small cell lung cancer, have shown responsiveness to immune checkpoint therapy, while tumors with low numbers of mutations are less likely to respond [182].

The success of immunotherapy based on CPI provides an opportunity to revisit vaccines as a potentially complementary mechanism of action to improve clinical efficacy when delivered in combination with CPI. The working hypothesis behind the CPI and cancer vaccine combination is based on the expectation that blocking the immunosuppressive TME would allow a cancer vaccine to boost already present anti-cancer T cells and induce ‘de novo’ T cell reactivities, thereby increasing the potency and breadth of anti-tumor immunity.

A major problem for the development of cancer vaccines is the se-lection of the appropriate tumor antigens. Since the identification of MAGE-1, the first tumor antigen which elicited an autologous cytotoxic T-lymphocyte (CTL) response in a melanoma patient [183], many other tumor antigens have been identified. The vast majority of previous cancer vaccines have targeted tumor-associated antigens (TAAs) that are self-antigens selectively expressed or overexpressed in tumors. TAAs include (i) cancer testis (CT) antigens normally expressed only in male germ cells, placenta or in early stages of embryonic development (e.g., MAGE-A1, MAGE-A3, and NY-ESO-1), (ii) differentiation antigens, mainly expressed on the tissue from which the tumor originated (e.g., gp100, tyrosinase, and Melan-A/MART-1), and (iii) antigens that are overexpressed in cancer cells (e.g. HER2, mesothelin, hTERT) [184]. All three TAA categories include autologous proteins against all of which immunological self-tolerance was established (though at different levels) during the ontogeny of the immune system. Thus, a cancer vac-cine based on TAAs must be able to “break tolerance” by stimulating the low affinity or rare TAA-reactive T cells.

Most recently, the development of next generation sequencing (NGS) led to the discovery of a novel category of tumor-specific antigens (TSA), the so called neoantigens, to which there is no self-tolerance [185].

Fig. 3. Overview of the process for generation of patient-specific NHPAd based neoantigen cancer vaccine. 1. The Patient has the tumor biopsy taken. 2. Tumor DNA and RNA and healthy tissue DNA (e.g., PBMC) are sequenced in order to identify the tumor specific mutations by comparing the sequences from normal versus tumor DNA. 3. A prioritization strategy is utilized to select the best neoAg for inclusion into the vaccine, based on 3 parameters: predicted HLA-I binding, allele frequency, mRNA expression. 4. The selected neoAgs are joined head to tail, with the mutated amino acid (aa), determined by a one to three nucleotides mutation, at the center and flanked upstream and downstream by 12 wild-type aa, for a total length of 25aa for each neoAg. The corresponding gene is cloned in a NHPAd vector. 5. The vaccine is produced by a rapid (few weeks) GMP manufacturing process.

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Neoantigens arise as a consequence of somatic mutations occurring in the coding regions of the cancer cell genome. The somatically mutated genes generate non-self-peptides which are processed and presented on MHC proteins on the surface of the malignant cells and subsequently recognized by autologous T cells as foreign antigens. Due to the lack of pre-existing central tolerance, neoantigens have the potential to be strongly immunogenic and capable of inducing potent and effective anti-tumor immunity. Cellular immunity directed against neoantigens was shown to be specific for mutant peptide sequences and does not cross-react with the wild type antigens [186], suggesting that the tar-geting of neoantigens is likely to be safe, with low risk of damaging healthy tissues. Therefore, given that neoantigens are 1) foreign to the body and 2) expressed only by cancer cells, they represent an ideal target

of a cancer vaccine. Most importantly, the relevance of neoantigens was established in the context of CPI treatment. In fact, a clear link was found between the success of CPI treatment and the number of mutations present in the tumor, the so called “tumor mutational burden” [182].

In summary, the field of cancer vaccine is currently facing a renais-sance arising from several key developments, including the clinical success of CPI, a better understanding of their role in cancer immunity and in modulating the activity of anti-cancer T cells together with the advent of NGS and the discovery and use of neoantigens as preferred targets of an immune attack against cancer.

Fig. 4. The multimodal mechanism of action of oncolytic viruses. (A) The injection of oncolytic viruses in primary/accessible tumors induces the oncolysis of cancerous cells, sparing healthy cells; the recruitment of immune cells within “cold” tumor microenvironment is promoted. Antiviral T cell immune response is supported, improving the lysis of OV-infected cells (1). The immunogenic way by which cancer cells suc-cumb, prompts the reinvigoration of antitumor immune response mediated by T and NK cells (2, 3). The presence of inflammation mediators within the tumor microenvironment promotes the polarization of tumor associated macro-phages to antitumor M1 phenotype (4). The acute inflammation and viral infection per se, upregulate immune checkpoints (i.e. PD-L1, PD-L2) supporting the combination of OVs with antagonist mAbs (5). The pre-existing im-munity to OVs, or the continual administration of the same viral species, induce antiviral hu-moral immune response that, in case of sys-temic delivery, can both inactivate the OVs (6) or upgrade the delivery to tumor site by anti-body dependent enhancement (ADE) of infec-tion (7). The elicitation of systemic antitumor immune response is also observed in non- injected tumor lesions that become infiltrated and may also regress (abscopal effect, 8). (B) Immunogenic cell death (ICD) is a core ac-tivity shared by different OVs. Depending on viral species, different mechanisms of immu-nogenic cell death can be induced. ICD leads to the activation of antiviral innate immunity (type I IFNs and interferon-stimulated genes), release of DAMPs, PAMPs, exposure of viral and tumor antigens. Tumor resident APCs can cap-ture these molecules and present antigens into the lymph nodes, activating both antiviral and antitumor T cells. The adaptive antiviral im-mune response can synergize with antitumor immune response both by helper T cells activity and by killing virus-infected cells. (C) The antitumor activity of OVs can be further enhanced by arming viral genomes with pay-loads. The cargoes can be: (1) pro-drugs acti-vators; (2) molecules for monitoring in vivo bio- distribution, or to allow in situ radiotherapy; (3) monoclonal antibodies targeting immune checkpoint modulators of tumor antigens; (4) cytokines (e.g. IL12, IL2, IL15, IFNs); (5) BiTEs targeting CAFs or tumor antigens; (6) tumor antigens; (7) extracellular matrix proteolytic cleavage; (8) ligands for co-stimulatory mole-cules (e.g. CD40 L, ICOSL, 4− 1BBL, OX40 L). Created with BioRender.com.

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5.2. Cancer vaccines targeting tumor neoantigens: preclinical and early clinical experience

Since the majority of cancer somatic mutations giving rise to neo-antigens are generated stochastically during cell duplication, every pa-tient acquires her/his own unique set of mutations (‘the mutanome’) that must first be identified for a rational vaccine design. The develop-ment of NGS techniques today allows a fast and comprehensive analysis of the tumor-specific mutations making it feasible the approach of personalized mutanome-based vaccines. Because many tumors accu-mulate hundreds of protein mutations it was necessary to develop al-gorithms capable of ‘selecting’ a subset of neoantigens with a higher probability of being present in the majority of the cancer cells present in the patient (represented by their allele frequency) as well as being capable of being presented in the context of the MHC I or II complexes (represented by their theoretical MHC binding affinity). A typical pro-cedure for identification of neoantigens is described in Fig. 3.

The NGS technology was applied for the first time to identify neo-antigens in mouse tumor models [187,188]. This led to the first pre-clinical demonstration of anti-tumor efficacy of a neoantigen based peptide vaccine in the murine melanoma B16F10 tumor model [188]. Similarly, vaccination with peptides reproducing the two neoantigens mLama4 and mAlg8 induced strong CD8 T cell responses and controlled tumor outgrowth of murine MCA sarcoma in both prophylactic and therapeutic settings with a degree of therapeutic protection comparable to that afforded by CPI [189]. An independent study also demonstrated that vaccination with mutated peptides identified in the murine MC38 tumor cell line by a combination of mass spectrometry, next generation sequencing and bioinformatics approaches was effective to induce tumor regression upon treatment [190]. A synthetic mRNA genetic vaccine encoding five neoantigens has been shown to be highly effective in a model of lung metastases [191].

While preclinical mouse studies using both peptides or RNA as vac-cine delivery systems have shown effective induction of neoantigen specific CD8 + T cell responses in mice, to date, the scenario appears quite different in patients. In 2017, two independent studies showed immunogenicity proof of concept of neoantigen-based vaccines in pa-tients by using the RNA and peptide delivery platforms. In both studies, vaccination boosted pre-existing neoantigen-specific T cell immunity and induced de novo neoantigen-specific T cells. However, the vaccines induced predominantly CD4 + T cells as assessed by ex-vivo IFN-γ ELISpot assay, while CD8 + T cells could be detected only after in vitro re-stimulation. This finding shed light on the choice of the vaccination platform for the induction of a potent CD8 + T cell response in humans [192,193].

5.3. The relevance of viral vectored vaccines for the induction of potent CD8 + T cell responses against many neoantigens

The induction of robust cellular immunity is an important require-ment to develop an effective cancer vaccine. The paradigm for an effi-cient induction of cytotoxic T cells is that the antigen is expressed from within the cell to be processed by the proteasome and be efficiently loaded onto MHC-I complex. As previously shown for genetic vaccines against infectious diseases, viral vectors represent an important delivery option. Viral vector infection not only allows for the expression of the antigen by antigen presenting cells, but also for the activation of the innate arm of the immune system and ultimately for a more effective induction of T cell immunity.

Several viral vectors have been evaluated in clinical studies as vac-cine targeting classical TAAs [194]. Compared to other viral vectors, replication incompetent NHPAd have the largest clinical casuistry of CD8 + T cell responses in humans (see paragraph 2 and Fig. 1). This becomes of relevance when considering that the induction of CD8 + T cell responses has been notoriously difficult to accomplish in cancer patients with the classical vaccination approaches .

Pre-existing immunity to the common HAd5 was shown to signifi-cantly blunt the immune responses induced by Ad5 vectored vaccines in humans and therefore, knowing that antibodies against Chimpanzee, Bonobo and especially Gorilla Adenoviruses have low/no seropreva-lence (0 %–18 %) in the human population, the NHPAds possibly represent the preferred Adenoviral vectors for the development of next generation cancer vaccines [195].

Tumors are extremely heterogeneous and NGS has revealed the presence of subclones of cells with distinct mutational patterns and divergent biologic behaviors [196]. Interactions between the immune system and tumors have clear functional significance for tumor control, as the immune system exerts evolutionary pressure on immunogenic tumor clones through the process of immunoediting [187]. Several different mechanisms can be put in place by the tumor to evade T cell attack including the loss of neoantigens at DNA level and reduced expression of neoantigen by gene promoter hypermethylation [197]. In this scenario, the ability of NHPAd vectors to accommodate large gene inserts and therefore to target many neoantigens, offers the unique op-portunity to potentially overcome the issue of tumor heterogeneity and escape through immunoediting. NHPAd vectors can express antigens of over 2200 amino acids [15] corresponding to a string of more than eighty 27 amino acid-long neoantigens.

Preclinical data recently published showed that immunization with a NHPAd vector encoding 31 neoantigens selected from the murine CT26 colon carcinoma cell line (referred to as GAd-31) was capable of inducing potent T-cell immunity, with overall >1000 IFNγ SFC/million splenocytes and the induction of both CD8+ and CD4 + T lymphocytes [198]. In line with other reports, only a fraction of the predicted neo-antigens was found to be immunogenic, highlighting the current limi-tations of the neoantigen prediction methods [199]. Also in this respect, the large number of neoantigens encoded by high capacity NHPAd offer the unique opportunity to potentially overcome the limits of prediction algorithms increasing the likelihood that at least a subset of the selected neoantigens included in the vaccine would be capable to induce an effective anti-tumor response. To date, the work by D’Alise et al. described the largest neoantigen based cancer vaccine and showed synergy between the GAd-31 vaccine and anti-PD1 leading to eradica-tion of large tumors in mice. Anti-tumor efficacy correlated with increased breadth and potency of neoantigen-specific T cells supporting the combination of neoantigen vaccine and CPI therapy for clinical studies [198].

A major obstacle for the clinical application of personalized vaccines is the need for a fast manufacturing and timely delivery of the individ-ually tailored vaccines for their administration to patients with advanced disease. The turnaround time for vaccine production depends on the type of vaccine format. In this respect, the NHPAd technology allows for a rapid (few weeks from neoantigen identification to filling in of the drug product) and reproducible production process for every set of neoantigens.

Currently, two NHPAd vectors are in clinical development for the delivery of neoantigen vaccines: a Chimpanzee Adenovirus (the species E ChAd68 serotype) and a Gorilla Adenovirus (species C GAd20 sero-type). Preliminary results in patients with advanced tumors, have demonstrated robust and consistent induction of CD8 + T cells against multiple neoantigens upon vaccination with ChAd68 (Granite trial, NCT03639714 and ESMO 2019). Therefore, the expectation for potent induction of ex-vivo detectable T cell immunity in humans against neoantigens by NHPAd vectors can be considered met.

The main limitation of vaccination approaches based on NHPAd vectors is the induction of anti-vector immunity after the first immuni-zation. Since several clinical studies have shown that heterologous prime/boost with different platforms elicits higher immune responses than repeated vaccination with an individual viral vector (see paragraph 3 of this Review) both the self amplifying RNA and MVA vector tech-nologies are currently being used to boost NHPAd vectors in cancer clinical trials (NCT03639714, ESMO 2019 and NCT04041310).

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The NHP/MVA prime/boost regimen with 2 viral vectors (GAd20 and MVA) is currently under investigation with a neoantigen based ‘off the shelf’ vaccine for Microsatellite Instable High (MSI-H) tumors (NCT04041310). The vaccine (Nous-209) is based on the concomitant administration of 4 viral vectors encoding overall 209 frameshift neo-antigen peptides shared among patients with MSI-H tumors [198,200]. Based on preclinical data, the NOUS-209 is expected to induce potent and broad CD8+ and CD4 + T cell responses in humans.

6. Oncolytic viruses: the endovaccines

The possibility to apply a vaccine strategy for the prevention of cancer has become one of the most effective measure in the field of oncology (e.g. human papilloma virus, HPV, vaccine). As detailed above, therapeutic cancer vaccines are more challenging as their activity could be hindered by consolidated immunosuppressive TME and large size of tumor burden. Besides the previously discussed personalized or off the shelf genetic vaccines, autologous or allogenic whole cell vacci-nation are under clinical development relying on the idea that cancer cells themselves effectively deliver the right tumor antigens [201]. Despite cell vaccination drives antitumor immune response, several clinical trials have demonstrated only a limited efficacy even if the formulations were adjuvanted by antigen presenting cells (APCs) or stimulating cytokines [202].

Oncolytic viruses (OVs) represent the next generation whole cancer cell endo-vaccine. Briefly, oncolytic viruses are naturally occurring or genetically engineered viral species able to selectively kill cancer cells without damaging healthy tissue (Fig. 4a).

To ensure tumor selectivity, different steps of the virus life cycle have been exploited (i.e.: entry, replication, transcription, translation). The most common mechanism of OV tumor selectivity is the attenuation of viral replication. This can be achieved by inactivation of viral genes whose function is not needed for replication in cancer cells, but it is essential for virus replication in healthy tissues (ie.: the ɣ34.5 gene of Herpes Simplex Virus which restricts replication in PKR or STING defi-cient tumor cells) [203]. Alternatively, replication defective OVs can be rescued by trans-complementation occurring only in tumor cells (i.e.: thymidine kinase inactivation in HSV or Poxviruses renders them dependent on endogenous levels of this enzyme, which is only found in high concentrations in dividing tumor cells) [204]. To maximise tumor cell lysis induced by of OVs, several strategies have been developed. The most common one relies on the isolation of new strains that are screened for antitumor activity and modified by introduction of attenuating mutations to provide tumor selectivity [205]. As an alternative to the attenuation of viral replication, and to retain the full replicative activity of the OV without compromising its safety profile, attempts have been made at modifying the virus tropism by engineering ligands specific for tumor-associated surface antigens (TAA) onto the membrane or the capsid of the virus particle [206–208]. This strategy provides for cancer selectivity by re-targeting the virus to the tumor cell and, in some cases, simultaneously enabling the de-targeting of the virus from its natural receptor (e.g. HVEM and Nectin1 for HSV-1). This approach is thought to be suitable for systemic delivery as it should minimize virus seques-tration and abortive replication in healthy tissues [342]. However, the impact of on-target, off-tumor toxicity due to the self-origin of TAAs still needs to be further evaluated in human clinical trials. Modulation of expression of genes essential for virus replication was also considered as a mean to increase tumor selectivity. For example, replacement of viral promoters with tumor associated promoters (ie.: TERT, Survivin, Nes-tin), or insertion of seed sequences in 3′UTR of viral transcripts targeted by miRNA downregulated in cancer cells led to cancer selective OV replication [209–212].

Because none of the methods described above is expected to avoid off target activity and with the aim of increasing tumor selectivity, com-binations of transcriptional conditioning and entry or translational re-striction have been developed potentially leading to improved safety

profiles [212,213]. Although up until very recently the mere killing of cancer cells

represented the cornerstone of oncovirotherapy, in the last decades, the way of thinking about the mechanism of action of OVs has dramatically changed and has transitioned to the dual ability of OVs to induce tumor cell lysis and immunologically reprogram the TME [214] (Fig. 4a). Tumor cells are notoriously non-immunogenic and they preserve this feature even during the daily homeostasis of mitosis and cell death. The immunotherapeutic activity of OVs depends on the “noisy” inflamma-tory way by which they destroy cancer cells, formally named Immuno-genic Cell Death (ICD). ICD is an evolutionarily-conserved way to detect non-physiological inflammatory processes including viral infections [215]. It consists of the release of damage- and pathogen-associated molecular patters (DAMPs, PAMPs), type I interferons, chemokines and “eat me” molecules. Oncolytic viruses induce ICD through distinct species-specific pathways including immunogenic apoptosis, nec-roptosis, pyroptosis and autophagy [216,217,341]. The highly immu-nogenic molecules associated to ICD recruit and activate several immune cell populations including APCs. APCs capture both viral and tumor antigens derived from oncolysis and become activated in the presence of ICD-derived molecules. Engulfed TAAs and neoAgs are delivered to draining lymph nodes where they are presented in the context on MHC molecules to T lymphocytes driving the maturation of tumor-reactive CTLs [218] (Fig. 4b). The antitumor immunity culmi-nates in the immune-infiltration of T cells, NK cells and other mediators of the adaptive immune response to the tumor lesions, thereby turning “cold” tumors into “hot” ones. This phenomenon is not limited to tumors with a preexisting anti-tumor infiltrate, but also for non-inflamed tu-mors defined as immune-excluded and immune-desert [219,220]. Even more interestingly, loco-regional non-injected and distal metastasis become infiltrated in a similar fashion to OV-injected tumor lesions, emphasizing the elicitation of an abscopal systemic antitumor immunity [221]. The activation of CTLs targeting TAAs (MART-1, IGFBP2 and FRα) has been demonstrated in patients treated with OncovexGM− CSF

and MV-NIS [222,223]. For these reason, OVs are now referred to as cancer endovaccines as they stimulate the anti-tumor adaptive response by exposing the immune system to endogenous TAAs and neoAgs which are produced in situ through OV mediated cancer cell killing.

6.1. Debated benefits of antiviral immunity

An intriguing and still debated aspect of oncolytic virotherapy is the role of antiviral immune response. In this regard, both innate and adaptive antiviral immunity could either increase or dampen the anti- tumor activity of OVs.

Innate antiviral immunity is a double-edged sword as Pattern Recognition Receptors engaged by PAMPs trigger type I IFNs produc-tion. IFNα/β dampen oncolysis as they are the most potent inhibitors of viral replication, but on the other side they are essential components of dendritic cell-driven T cell responses to cancer [224–227]. The activity of Stimulator of interferon genes protein (STING) recapitulates well the concept of antitumor benefit of antiviral immunity. Its tumor intrinsic activity has recently been described as essential to both undermine the replication of oncolytic virus and to elicit the OV-mediated cancer vaccine through induction of immunogenic cell death [341].

Recent evidences suggest that many proteins of different viral spe-cies, have evolved to escape the antiviral mechanisms of the host by overcoming type I IFNs restriction. Most of these molecular machineries utilized by viral proteins inhibit cGAS and/or STING protein to prevent the sensing of viral genomes and the downstream activation of inter-feron related genes [228]. The consensus of opinion in oncovirotherapy field is that preserving the viral property to escape from the host innate response would provide a better anti-tumoral activity [207,211,212, 228–231].

As far as adaptive anti-viral immunity is concerned, no immuno-logical interference has been observed between anti-viral and anti-

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tumor adaptive immunity. Anti-viral specific T cells have been reported within the tumor immune infiltrate upon OV treatment and, in some cases, anti-tumor immunity seems not to be affected even in case of preexisting anti-viral immunity [232,233]. The lack of interference on anti-viral immunity by cancer cells is possibly due to viral antigens being presented to naïve or experienced, but not exhausted, T lymphocytes, and to the transient nature of viral antigens as opposed to the persistence of the cancer ones [203,234,235]. On the other end, anti-tumor immu-nity might be reinforced by the presence of infiltrating anti-viral T lymphocytes, this effect possibly due to anti-viral CD4+ helper T cells that can activate antitumor CD8 + T cells in a shared immunological synapse [236,237] (Fig. 4). Although the innate and adaptive anti-viral immunity are likely to have a positive impact in case of intratumor administration of OVs, the influence of humoral anti-viral response is more challenging for systemic delivery. Indeed, except for the uncom-mon Antibody-Dependent Enhancement (ADE) of viral infection re-ported for oncolytic reovirus (Reolysin) and Coxsackievirus type A21 (CVA21, CAVATAK), the presence of neutralizing antibodies could interfere with the systemic delivery of OVs [238,239] (Fig. 4a). Thus, the complexity of antiviral immunity in the context of oncolytic viro-therapy is far from being elucidated and its contribution to the efficacy of OV treatment will probably depend on the viral species adopted and the tumor indication.

6.2. Priming tumor microenvironment to immune checkpoint inhibitors

As pronounced immune infiltrate phenotype of tumors predicts a favorable response to immunotherapies, the combination of OVs and immune checkpoint inhibitors was immediately considered. This com-bination has been further supported as OV-induced immune infiltration is often accompanied by up-regulation of immune checkpoint molecules [240,241] (Fig. 4a). Many preclinical studies demonstrated the synergy of CPI to different OVs, including Herpes virus (HSV), Vaccinia virus (VV), Adenovirus, Myxoma virus, Reovirus, Measles virus, Vesicular stomatitis virus (VSV), Maraba virus, Newcastle disease virus (NDV), Coxsackievirus, Poliovirus and Yellow fever virus [242–246]. This syn-ergy is exemplified by the broadening of TAA and neoantigen-specific T cell reactivities and improved survival following combined treatment with OVs and CPI in mouse tumor models [247,248]. The evidences collected in preclinical settings supported the clinical use of the com-bination of PD-1 and CTLA-4 blockade with HSV OV Talimogene laherparepvec (T-VEC; Imlygic) and other OVs as recently reviewed [244,249,250]. The first randomized trial testing the combination of OV and CPI was applied to melanoma patients with T-VEC plus CTLA4 blockade (Ipilimumab). At the three-years follow up, the combination group demonstrated enhanced objective response rate (ORR) (37 % combo vs 16 % Ipilimumab) and complete response rate (21,5% combo vs 6% Ipilimumab) (NCT01740297). The superior efficacy of the com-bination was not limited to injected lesions, as locoregional and visceral metastasis were also decreased [251]. To date, thousands of patients have been enrolled in OV/CPI combination studies carried out with Herpesviruses (T-VEC, HF10, RP1, RP2), Adenoviruses (Tasadenoturev, ONCOS-102, Enadenotucirev, VCN-01, LOAd703, ColoAd1, OBP-301, CG0070), Vaccinia virus (Pexa-vec), VSV (VSV-IFNβ-NIS), Maraba virus (MG1), Measles virus (MV-NIS), Reovirus (Reolysin Pelareorep), Poliovirus (PVS-RIPO), Coxsackievirus (CVA21 CAVATAK) and NDV (MEDI5395) [244,252–254]. The growing clinical experience is under-lining how OV treatment immunologically primes the patient’s TME to immune checkpoints, making OV/CPI combination an encouraging strategy to overcome intrinsic and acquired resistance to immune checkpoint blockade [240,255–257]. One of the most relevant aspects of oncovirotherapy is the long persistence of the antitumor immune response that has been well documented by protection against a second tumor challenge in preclinical models [207,212,258]. This feature could be exploited in a next future to provide patients at high risk of tumor recurrence with an antitumor immunological memory to prevent the

relapse of disease. The efficacy of this approach has been demonstrated in a pre-surgical preclinical setting underlining the possibility to use OVs as first-line therapeutic agent in combination with established clinical approaches including surgery [259,260].

6.3. Integration of oncolytic virus and intra-tumoral gene therapy

For the purpose of potentiating in situ vaccination by OVs, the expression of therapeutic proteins encoded by OVs has been widely exploited in pre-clinical and clinical experimentation. This approach allows to integrate oncolysis to gene therapy, turning cancer cells in drug factories before their death. Many of the tested payloads are pleiotropic genes with multimodal mechanism of action, but for ease of reference we have classified them in: i) cytotoxicity boosters; ii) medi-ators of tumor biostructure remodeling; iii) tumor antigens and neo- antigens; iv) immune modulators (Fig. 4c).

6.3.1. Cytotoxicity boosters The reduction of tumor bulk is an essential facet of oncovirotherapy.

The intratumoral viral load decreases over time, eventually disappear-ing, due to innate and adaptive anti-viral immunity. For this reason, it is nowadays assumed that oncolysis alone is not sufficient to completely destroy the tumor bulk. To selectively improve the cytotoxicity within the tumor environment, OVs have been armed with cell suicide genes able to convert non-toxic compounds into cytotoxic drugs [261] (Fig. 4c). The most common suicide genes are Herpes simplex virus thymidine kinase (HSV-TK), yeast cytosine deaminase (CD) and the chimeric enzyme FCU1 (cytosine deaminase FCY1 fused to uracil phosphoribosyl transferase FUR1 protein), that are able to metabolize ganciclovir and 5-fluorocytosine (5-FC) pro-drugs, respectively [262–266]. One of the most representative suicide gene-armed OV is the investigational Toca 511 (vocimagene amiretrorepvec), a tumor-selective non-lytic retroviral replicating vector expressing CD to convert Toca FC (an extended-release formulation of flucytosine) into the cytotoxic chemotherapeutic 5-fluorouracil (5-FU) [267,268]. The combination of Toca 511 and Toca FC was implemented for patients with recurrent brain tumors including high grade glioma and glioblas-toma (NCT04105374, NCT01156584, NCT01470794, NCT02414165, NCT01985256). A similar approach is radio-virotherapy that consists of arming OVs with a transmembrane transporter (e.g. sodium-iodide symporter, NIS) to concentrate radiopharmaceuticals within the tumor allowing both the tracking of viral spread and in situ radiotherapy. Ongoing clinical trials are investigating the safety and preliminary anti-tumor activity of NIS encoding OVs (VSV-IFNβ-NIS, Voyager V1: NCT03647163, NCT04291105; MV-NIS: NCT03171493, NCT02364713) [269,270]. A completely different approach to enhance viral oncolysis relies on the formation of syncytia. Formation of syncytia allows viral particles to spread from a single infected cell to non-infected bystander ones increasing both the lysis of the tumor mass and the immunogenicity of these multinucleated giant cells. The formation of syncytia is a naturally occurring feature of many viruses (MV, NDV), but fusion-enhanced oncolytics have been generated by encoding fusogenic proteins within their viral genome. RP1 is a herpes simplex virus type 1, ‘armed’ with two genes: a gene encoding gibbon ape leukemia virus fusogenic protein (GALV) which was shown to increase immunogenic cell death, and a gene encoding GM-CSF to increase the immune/in-flammatory cascade (NCT04336241, NCT04050436) [205,271–277].

6.3.2. Mediators of tumor biostructure remodeling The high density of tumor cells and stromal components (i.e.: tumor

vasculature and extracellular matrix) modulates acidity, O2 and nutrient availability as well as the delivery of drugs and the access of immune cells to the tumor which is hampered by the tumor interstitial pressure [278]. Cancer associated fibroblasts (CAFs) are key players in orches-trating the deposition of extracellular matrix components [279]. Onco-lytic herpesvirus and adenovirus able to efficiently degrade components

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of the extracellular matrix and enhance viral spread and drug delivery by encoding metalloproteinase 9 (MMP9) and hyaluronidase within viral genome, respectively, were generated, with the latter one (VCN-01) showing to be safe and to induce intravitreous infiltration of CD4+ and CD8 + T cells in patients with refractory retinoblastoma [280–283] (Fig. 4c).

To act upstream of the extracellular matrix deposition and destroy CAFs, oncolytic Adenovirus T-SIGn has been armed with a Bispecific T- cell Engager (BiTE) to crosslink T cells (via the T cell receptor compo-nent CD3ε) to cancer associated fibroblasts (via fibroblast activation protein, FAP). In a preclinical model, a FAP-BiTE–encoding virus induced fibroblast death and T-cell activation, paving the way for clin-ical assessment (NCT04053283) [284]. The second approach for TME remodeling relies on OVs armed with anti-angiogenic or vasculostatic agents to deprive cancer cells of blood supply required for nutrient and oxygen support [285,286].

6.3.3. Tumor antigens and neoantigens In situ vaccination induced by the release of tumor associated anti-

gens and neoAgs can be strengthened by shuttling the epitopes of in-terest by OVs (Fig. 4c). Oncolytic measles virus and Maraba virus MG1 have been used as genetic shuttles of TAAs. In several preclinical models it has been demonstrated their ability to engage effector immune cells targeting the OV-encoded TAAs (MAGE-A3, Claudin6, HPV E6 and E7 proteins) [287–290]. In particular, an heterologous prime/boost regimen based on non-replicating Adenovirus combined to oncolytic Maraba virus MG1, both expressing MAGE-A3, has been proved to induce the expansion and long-term persistence of TAA-specific immune response in Macaca fascicularis. Currently, this regimen is being eval-uated in phase I/II clinical trials as monotherapy and in combination with PD-1 inhibition (NCT02285816, NCT02879760) [291]. Because of the self-origin of TAAs, their recognition by CTLs can be limited by central tolerance (except for oncogenic virus antigens) and could be responsible of on-target, off-tumor toxicity. Moreover, especially in case of TAAs that are not essential for cancer cell survival and proliferation, the immune pressure of T cells can silence the expression of TAA and make tumor cells “invisible” to CTLs. To circumvent these limitation MY-NEOVAX, an oncolytic adenoviral platform encoding up to 50 pa-tients’ specific neoAgs was developed. As discussed in paragraph 5 of this Review the neoAg-based personalized approach has the potential to circumvent the immune escape mechanisms due to epitope loss. In addition, as described above, the cytolytic activity of the OVs stimulates the release of molecules (cytokines and chemokines) that recruit and activate anti-tumor effector T cells counteracting the effect of TME immunosuppression. While still limited to a small number of cases, the MY-NEOVAX therapy proved to be effective in improving survival in two patients with last-line treatment refractory colorectal cancer and high-grade neuroendocrine carcinoma [292]. At variance with vectors encoding tumor antigens, PeptiCRAd and PeptiENV are oncolytic viruses coated with peptides reproducing tumor-specific epitopes. In preclinical models the delivery of tumor peptides carried by oncolytic viruses showed the ability to enhance the immunogenicity of tumors and improve immune infiltration, sensitizing poorly immunogenic tumors to checkpoint blockade [293–295]. The versatility of this platform could allow to “dress-up” oncolytic virus with patient-specific neoAg peptides for translation into the clinic.

6.3.4. Immune modulators Cytokines regulate nearly every immune-related process and inte-

grate all the compartments of the immune system through the polari-zation, activation and recruitment of leucocytes. The discovery of antitumor activity of some these molecules (IL-2, IFNs) represents a cornerstone of cancer immunotherapy research. Despite the great ex-pectations raised in preclinical models, the translation into the clinic demonstrated only mild clinical benefit due to short half-life, insuffi-cient concentrations in the tumor and severe adverse events due to

systemic administration and high dosage [296]. To circumvent these issues, in situ production of cytokines by intratumoral delivery of cytokine encoding OVs is a promising approach (Fig. 4c). At present, several OVs under pre-clinical and clinical investigation are armed with native or modified forms of cytokines including, but not limited to, GM-CSF, IL-12, IL-2, IL-15, IL-18 and IFNs (type I and II) [207,297–301]. Compared to ‘naked’ OVs, the corresponding cytokine armed ones enhanced proinflammatory signals and the infiltration of CD8 + T and NK cells improving the ratio of cytotoxic/regulatory T cells. With the approval of T-VEC (OncovexGM− CSF), Granulocyte-macrophage colo-ny-stimulating factor (GM-CSF) has been widely adopted as cargo in many clinical oncolytic viruses including Replimune’s RP viruses (NCT04336241, NCT04050436), ONCOS-102 (NCT02963831, NCT03514836), Pexa-Vec (JX-594, NCT02562755) and CG0070 (NCT01438112, NCT04387461) [205,302–306]. Besides GM-CSF, the IL-2 armed TILT-123 adenovirus (NCT04217473) and IL-12 encoding HSV-1 (M032, NCT02062827) and Adenovirus (Ad5-yCD/mutTKSR39-rep-hIL12, NCT03281382) were also advanced into the clinic [307–311]. At the same time, efforts are still dedicated at improving the effectiveness of the encoded cytokines through the engineering of chimeric proteins such as the super-agonist IL-15 (IL-15_IL-15Ralpha) and at reducing systemic adverse effects by confining the cytokines to the TME by molecular anchors [312–317].

Immune checkpoint modulators, both costimulatory and inhibitory have become the main arm of cancer immunotherapy. The combination of checkpoint blockers (e.g. PD-1 and CTLA-4; NCT01844505) was effective at increasing the response and survival rates in multiple cancer indications, however, the incidence of severe immune-related adverse events was also enhanced (from 16 %–27 % in patients receiving mon-otherapy to 55 % in the combination group) [318,319]. In addition, the cost of antibody-based immunotherapy related to manufacturing pro-cesses and the need for repeated administrations led to the adoption of OVs as gene therapy carriers for CPI and co-stimulatory ligands poten-tially allowing higher concentrations at the tumor site and draining lymph nodes while decreasing systemic administration and side effects [320,321]. Anti CTLA-4 antibodies and PD-1/PD-L1 inhibitory agents have been delivered by several OVs in preclinical models demonstrating similar efficacy to the combination of unarmed OVs plus systemic administration of checkpoint blocking antibodies [205,248,298, 322–327]. The HSV-1 RP2 oncolytic virus is based on the oncolytic RP1 HSV1 encoding GM-CSF, GALV and an anti-CTLA-4 antibody, and it is now being tested in a first-in-human phase I as monotherapy and in combination with PD-1 blockade in patients with injectable solid tumors (NCT04336241).

As a consequence of oncolytic viral infection and ‘warm up’ of the tissue microenvironment, the display of co-stimulatory receptors may also increase the immune cell populations. However, the upregulation of activation receptors (ICOS, OX40, 4-1BB, CD40, CD28, GITR) is not often accompanied by a corresponding increase of the cognate ligand to optimally trigger T cells and APCs activation. Providing in trans the receptor-matched ligands by OVs has been validated for many targets in preclinical models (OX40 L, 4-1BBL, ICOSL, CD40 L, CD80, GITRL) and in clinical trials demonstrating the enhancement of antitumor immunity without evidences of adverse effects (OX40 L by DNATrix’s DNX-2440, NCT03714334; CD40 L and 4-1BBL by Lokon Pharma’s LOAd703, NCT02705196, NCT03225989, NCT04123470; CD40 agonist by PsiOxus’ NG-350A, NCT03852511; CD80 plus anti CD3 engager by PsiOxus’ NG-348, Licensed to Bristol-Myers Squibb) [205,297, 328–333]. Moving away from the use of canonical immune mediators, the clearance of immunosuppressive molecules from the TME has also been considered. Prostaglandin E2 (PGE2) exerts various pathological processes in cancer, including cancerous cell proliferation and invasion as well as expansion of myeloid derived suppressive cells (MDSCs) and inhibitory T regulatory cells (Treg) [334]. To reduce intratumor PGE2 levels, an oncolytic vaccinia virus has been engineered to express prostaglandin-inactivating enzyme hydroxyprostaglandin

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dehydrogenase 15-(NAD) (HPGD). The expression of HPGD selectively depleted Treg and MDSCs populations, thereby enhancing the antitumor immune response by upregulation of Th1-associated chemokines (e.g. CXCL10, CCL5) [335]. Similarly, a metabolic-reprogramming of TME has been achieved by engineering oncolytic vaccinia virus to express leptin. Through a set of biochemical assays, the authors demonstrated that the delivery of leptin, in the context of OV-induced immune infil-tration, improved mitochondrial oxidative phosphorylation (OXPHOS) in tumor infiltrating lymphocytes, preventing CD8 + T cells metabolic exhaustion and thereby enhancing therapeutic efficacy and antitumor memory development [336,337].

Preclinical and clinical evidences collected in last decades have shed new light on the mechanism of action of OVs. The lack of correlation between viral replication or cell killing and in vivo antitumor efficacy suggested that most of the therapeutic activity of OVs is mediated by the induction of adaptive antitumor immunity and in situ vaccination, but many aspects are still unclear [214,338]. Gathering more clinical knowledge from the T-VEC experience, along with the growing number of phase I to III clinical studies with OVs and CPI combinations will help better understanding how to optimize the use of these viruses for cancer therapy. The stratification of responder and non-responder patients ac-cording to biological or genetic markers could in the next-future support the identification of those patients that are more likely to achieve ben-efits from oncovirotherapy. Finally, continued technological improve-ments may allow the safe administration of OVs by a systemic route thus broadening their range of application. Along this line a promising way to provide the best outcome to all patients is to supply immune potenti-ating factors as payloads within the viral genome.

7. discussion/final remarks

Early vaccines were based on whole inactivated or live attenuated microorganisms such as those against smallpox and poliovirus that led to worldwide eradication or restriction of these infections [339]. The high effectiveness of these vaccines was due to their ability to recapitulate the wide spectrum of immune responses induced by the pathogen itself and including both arms of the adaptive immunity as well as innate immunity.

Lately, to increase safety, vaccine technologies transitioned to sub-unit vaccines, some of which were shown to be extremely effective such as those against HBV and HPV [340], but in doing so, something got lost in the ability to induce a full spectrum of immune responses such as cellular immunity and in particular cytotoxic T cells. Thus, this second generation vaccines are less suitable for preventing/curing some infec-tious diseases for which humoral immunity alone may not be sufficient or even ineffective such as HIV, Malaria, Tuberculosis, and above all

cancer. To rescue the ability to induce both arms of the adaptive immunity,

third generation genetic vaccines and in particular viral vectored vac-cines based on non-pathogenic or replication incompetent viruses, have come on stage due to their mechanism of immune stimulation closely mirroring that of the pathogen itself while presenting a safer profile. While highly promising, however, viral vectors have not provided for the ultimate solution to all vaccine applications. In fact, as of today, several viral vector platforms have successfully reached the clinical stage, but only Ervebo, the Ebola Zaire VSV-based vaccine, was approved for the prevention of Ebola virus disease (EVD) caused by Zaire ebolavirus in individuals 18 years of age and older. This is mainly due to each viral vector presenting with different characteristics which render them more or less suitable for a specific vaccine application. Thus, in search for novel more effective vectored vaccines, the rational approach based on protection studies in animals and by inference from immune responses shown to protect against repeated natural infection (the so-called mechanistic correlates of protection) must be adopted to increase probability of success.

In this review we have provided an outlook of the most recent results obtained preclinically and clinically with a selected number of viral vectors, highlighting their most relevant features (see Table 1) such as the type of immune responses induced, the ability to be re-administered in homologous prime/boost regimens, the capacity to encode large an-tigens and the ability to be produced on a large scale. This was done with the specific objective of describing a potential ‘toolbox’ of different technologies that can be interrogated while looking at specific solutions to different vaccination needs.

Funding

This work was supported by the Grants from "SATIN, Regione Campania" and "CEINGE TASK-FORCE COVID-19", Regione Campania

Declaration of Competing interest

Emanuele Sasso is an employee of Nouscom srl and a co-author of patent applications on oncolytic viruses. Anna Morena D’Alise is an employee of Nouscom srl and a co-author of patent applications on NHPAd based vaccines and oncolytic viruses. Elisa Scarselli is an employee of Nouscom srl, a shareholder of Nouscom AG, and a co- author of patents and patent applications on NHPAd based vaccines and oncolytic viruses. Antonella Folgori is an employee of Reithera srl, a shareholder of Keires AG and Nouscom AG and a co-author on NHPAd based vaccines. Alfredo Nicosia is a shareholder of Keires AG and Nouscom AG and a co-author of patents and patent applications on

Table 1 Characteristics of viral vectors used for vaccines against infectious diseases.

Vector System Replication Competent*

Integration Insert Size

Antibody** T CD4** T CD8** Short term readministration

Manufacturing cells

Clinical stage

POXVIRUSES (MVA)

No No 7.5 Kb Low/ Moderate

Low No Yes CEF, AGE1

Phase III^

ADENOVIRUSES (NHPAd)

No No 7.5 Kb Moderate High High Yes^^ PERC6, HEK293, Procell 92

Phase III

RHABDOVIRUSES (VSV)

Yes No 6 Kb Moderate/ High

Low/ Moderate

Low/ Moderate

Yes Vero Approved

PARAMIXOVIRUSES (MV)

Yes No 6 Kb High NA NA Yes Vero Phase II

ARENAVIRUSES (LCMV)

No No 2 Kb Moderate/ High

Moderate/ High

Moderate/ High

Yes HEK293-GP Phase I

HERPESVIRUSES (HCMV)‡

No§ No 6 Kb High High High Yes MRC5 Phase I

Antibody: induction of antigen-specific antibodies in humans; T CD4, induction of antigen-specific CD4 + T cells in humans; T CD8: induction of antigen-specific CD8 +T cells in humans; * refers to vaccine vectors used in clinical studies; ** refers to clinical studies; ̂ refers to recent clinical studies where MVA was used to boost natural or artificially induced primary response;^^ only for antibodies; ‡ used as an HCMV vaccine with no heterologous antigen cargo; § highly attenuated; NA = clinical data not available.

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NHPAd based vaccines and oncolytic viruses. Nicola Zambrano has no competitive interests to declare.

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