Coronaviruses (CoVs) are RNA viruses that belong to the family Coronaviridae in the order Nidovirales. CoVs are divided into 4 major genera: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus [1,2]. They have a broad host range, including humans, birds, bats, camels, and other mammals [3]. CoV infections can result in respiratory, gastrointestinal, hepatic, or neurological diseases with mild to severe severity. Although some animal CoVs are species-specific, others can cross species barriers, potentially leading to zoonotic spillovers to humans [4].

CoVs are single-stranded RNA viruses with a positive-sense genome ranging from 26 to 32 kb [5-7]. Their genome is organized so that the 5’ two-thirds encodes the replicase genes, while the remaining one-third at the 3’ end encodes structural genes [8-10]. Approximately 20 to 22 kb of the genome is dedicated to the replicase genes, which include open reading frame (ORF) 1a and ORF1b [9,11]. The ORF1a region encodes papain-like proteases and picornavirus 3C-like proteases, while ORF1b encodes viral RNA-dependent RNA polymerase, helicase, and exoribonuclease [11]. Translation begins at ORF1a and extends to ORF1b, producing polyproteins that are then cleaved by viral proteases into 16 mature non-structural proteins. Proteins encoded by ORF1b are generated in relatively smaller quantities than those from ORF1a because of the 25% to 30% frequency of ribosomal frameshifting. Despite this, ORF1b is the most conserved region of the CoV genome and plays a critical role in viral replication [12,13]. The 3’ one-third of the genome encodes major structural proteins, including spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins, as well as accessory proteins with partially understood functions. The S, E, and M proteins are located on the surface of the virion, with some CoVs also expressing the hemagglutinin esterase protein [6].

The development of CoV vaccines has been a critical area of focus, particularly during the coronavirus disease 2019 (COVID-19) pandemic, with efforts to prevent the spread of the virus and reduce the disease severity [14]. Vaccine development typically targets the S protein of CoVs because this protein plays a central role in viral attachment and entry into host cells. The receptor-binding domain (RBD) of the S protein is especially significant because it mediates binding to host cell receptors like angiotensin-converting enzyme 2 (ACE2) in the case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [15,16]. Vaccines can effectively neutralize the virus and prevent infection by eliciting an immune response against the S protein [16]. Modern vaccine platforms, such as mRNA-based vaccines, viral vector vaccines, protein subunit vaccines, and inactivated or live-attenuated virus vaccines, have been used for CoV vaccine development [17-19]. Each vaccine platform has distinct advantages and challenges. For example, mRNA vaccines can be developed rapidly but require cold storage [20], whereas viral vector vaccines are more stable but may face pre-existing immunity to the viral carrier [21]. Despite these challenges, vaccines have been highly effective in reducing the severity of CoV infections and controlling the spread of the virus [22].

Adenoviruses have many advantages over other viral vectors, such as the vaccinia virus, lentivirus, retrovirus, adeno-associated virus (AAV), and herpesvirus, because of their ability to induce strong adaptive and innate immune responses in the host [23]. For example, lentiviruses and retroviruses integrate their viral DNA into the host genome, which can lead to mutagenesis. Adenoviruses, however, avoid this risk because they do not integrate their genome into the host DNA. Although AAV is less pathogenic [24] than adenoviruses, its large-scale production is more complex and challenging [23]. This review describes the biological features of adenovirus-based CoV vaccines, particularly the current efforts focusing on vaccines targeting human CoVs. This review outlines the impact and development of adenovirus vaccines that have been majorly trialed on animals.

Adenoviruses are non-enveloped viruses with an icosahedral nucleocapsid containing double-stranded linear, non-segmented DNA of 26 to 45 kb in size, depending on the serotype [25]. Human adenovirus type 5 (Ad5), approximately 36 kb, is the most widely used adenovirus serotype [26]. The viral genome is flanked by inverted terminal repeats (ITR) at both ends. The packaging signal (ψ) at the left arm of the genome is required for the proper viral transcript packaging. The viral transcripts are in 2 forms: early and late. The 4 early transcripts, E1, E2, E3, and E4, are responsible for regulatory functions, including expressing non-structural proteins. The late genes encode the structural genes (Fig. 1A) [27]. The transgene capacity of the adenoviral vector (AdV) has been improved through several generations of vector construction [28]. The first-generation AdV was constructed by deleting E1A and E1B regulatory genes [29]. In addition to the E1 gene, E2, E3, and E4 non-structural genes are deprived in the second-generation AdV to increase the cloning capacity and decrease the viral replication capacity. On the other hand, the second-generation AdV triggered immune responses against the AdV itself in vivo. Therefore, the third-generation AdV was constructed to be less immunogenic in that the vector consists only of ITRs and packaging signals, stripping all coding sequences. Hence, the third-generation AdVs are called high-capacity AdVs or gutless AdVs (Fig. 1B) [30,31].

Adenoviruses are powerful adjuvants because they stimulate immune responses [32]. In addition to the immunogenic capacity of adenovirus vectors, the viral genome can be modified by inserting exogenous transgenes of interest [33]. Furthermore, adenoviruses can activate numerous innate immune signaling pathways that induce the secretion of inflammatory cytokines. Therefore, adenoviruses are potent immune activators that attract antigen-presenting cells to the infection site, increase T-cell priming, and enhance inflammatory responses. These properties of adenoviruses to activate the innate and adaptive immune responses generate potent immunity against exogenous antigens [34].

Adenovirus-based vaccines produce long-lasting responses, but the immune responses generated by adenovirus-based vaccines decline over a shorter period and remain at low levels for a longer period [4,35]. They have a wider range of effective doses, and most importantly, they do not require sub-zero temperatures for storage and can even be kept at environmental temperatures for days. This makes the adenovirus-based vaccines preferable when it is difficult to maintain the cold chain and are less expensive than mRNA vaccines [36].

Adenovirus-based vaccines generate a phenomenal level of CD8+ T-cell responses, in addition to the specific antibody responses in various animal models [37]. The immune responses can have variations depending on the serotype involved. The importance of these CD8+ T cells is that they are long-lived, and the partially activated CD8+ T cells initiate effector functions immediately, unlike memory B cells, which take several days to commence the effector functions [38]. Although the CD8+ T cells cannot hamper the infection, they kill the pathogenic cells immediately after infection, preventing new progeny virion production and spreading, shortening the infection, lessening the severity, and decreasing the transmission [36].

Adenoviruses are versatile and diverse; they are grouped into 7 subtypes: A-G and 67 serotypes. This stratification relies on the similarities in genome homology, tropism, and analysis of neutralizing antibody (nAb) against capsid antigen, the hexon structure, and viral DNA polymerase. Apart from these advantages, adenoviruses have some weak spots as vaccine adjutants. This drawback can be overcome by replacing the adenovirus hexon sequence or the fiber knob domain (chimeric adenovirus) with a different serotype [39]. The Ad5 vector is closely associated with the human population. Thus, the vector immunity is hampered by pre-existing immunity [40]. The less seroprevalence adeno-serotypes have been examined to mitigate the pre-existing immunity of the Ad5 vector [41-43]. Ad5 is used frequently in adeno-based vaccine studies because it is more efficacious than the other serotypes (Ad11, Ad35, Ad50, Ad26, Ad48, and Ad49) [44]. In addition, introducing a calcium phosphate (CaP) artificial shell on the virus surface is an effective way to escape the pre-existing-immunity [42,45]. Combining different adenovirus serotypes is possible because the nAbs and T cells produced from different serotypes do not cross-react. Therefore, the immunostimulatory properties and their well-characterized safe profile make the adenovirus vectors feasible for use as virus vectors to elicit effective and strong immune responses for target antigens [46,47].

Adenovirus vector-induced thrombotic thrombocytopenia syndrome (TTS) usually occurs 21 days after vaccination. In the United States, 60 cases of TTS and 9 deaths were reported for 194 million vaccine doses administered. Nordic and Asian countries reported 17.6 cases per million doses, while Brazil reported 0.2 cases per million doses. The precise etiology of vaccine-induced TTS is still unknown. Patients with TTS develop thrombocytopenia and autoantibodies to platelet factor 4 (PF4) [48]. Some test-tube studies believe that the autoantibodies arise from complexes formed between the negatively charged PF4 and positively charged adenovirus hexon. This explanation is dispelled by the fact that Ad26 and Ad5 of Sputnik V do not cause TTS. This rare occurrence may be explained by differences in the immune status and genetic composition of those given the vaccines. These side effects could be attributed to the interaction between the adenovirus and the SARS-CoV-2 S protein, which acts as a super antigen and causes a cytokine storm [49].

Ten adenovirus-based CoV vaccines have been studied for their efficacy; 4 have been approved for human use: ChAdOx1-nCoV-19 (AZD1222) developed by Oxford/AstraZeneca, Ad26.COV2-S (Jcovden) by Janssen/Johnson & Johnson, Gam-COVID-Vac (Sputnik V) by the Gamaleya Research Institute, and AD5-nCOV (Convidecia) by CanSino Biologics [19]. These vaccines use Ad5, Ad26, and a chimpanzee adenovirus serotype. Although rare cases of vaccine-induced TTS have been reported, these vaccines have shown efficacy in combating SARS-CoV-2 and remain in active use [50].

Table 1 lists the progress of research on adenovirus-based SARS-CoV vaccines. The development of adenovirus-based SARS-CoV vaccination deals with identifying the most suitable antigenic fragment or combinations and the administration route to elicit the highly efficacious and safe vaccination regimen. The S protein of SARS-CoV has been used in its full-length form [51-53] and truncated S1 fragment [54]. The sera from convalescent SARS-CoV patients recognize the antigenic epitopes on the S protein and can induce nAbs [54,55]. The N protein is the most abundantly expressed immunogen in SARS-CoV infections [2] and the most important diagnostic antigen of SARS-CoV [56].

Nevertheless, some antigenic dominants are not immunodominant in SARS-CoV patients but induce antibody responses in immunized small animals [2]. Recent studies have highlighted the humoral and cellular immunogenic capacity of the SARS-CoV N antigen coded to adenoviruses [2,56-59]. The M and E proteins are also important in protection [56]. Combinations of S, N, and M proteins have been tested to induce high concentrations of virus-specific antibodies in CoVs [60,61] and adenovirus-based SARS-CoV vaccines [57] because the S1 protein is most likely to generate neutralizing responses, and the N protein is a delegate antigen for T-cell responses [60]. The intramuscular (IM) administration of adenovirus-based SARS-CoV vaccines is frequently practiced [62]. The intranasal (IN) administration of the adenovirus-based SARS-CoV vaccines has been proven to be efficacious in eliciting antibody responses because adenoviruses naturally infect the mucosal sites, and the primary infection site of the respiratory viruses is dealt with mucosae [57]. On the other hand, the retrograde transport of the vaccine to the olfactory bulb [62] is a concern for this alternative admission route, like sublingual (SL) immunization [51]. Despite this, adenovirus-vectored vaccines via the IN route exhibit little or no viral dissemination in a major region of the central nervous system, the brain [63]. SARS-CoV is associated with hepatitis in humans [64].

The S protein in modified vaccinia Ankara (rMVA-S) immunization in ferrets also reported hepatitis after a challenge with SARS-CoV, but it was not demonstrated that it was a consequence of viral infection or a combined effect of a rMVA-S and SARS-CoV infection [52]. Nevertheless, adenovirus-vectored SARS-CoV-S did not induce any liver cytotoxicity in ferrets. This may be attributed to the non-replicating adenovirus vector or the meaningful protection from the heterologous adenovector combination (AdHu5 and AdC7) [53]. The pre-existing immunity for the adenoviruses is a concern for adenovirus-based vaccines. The desire for strong humoral and cellular antigenic-specific immune responses can be achieved using a heterologous combination, priming with DNA vaccines [59]. In contrast, animals pre-exposed to the Ad5 vector still responded the same way as non-exposed animals, highlighting the potential use of the adenovirus vector platform in people even if they had a pre-exposure [60].

Table 2 lists the progress of research on adenovirus-based Middle East respiratory syndrome coronavirus (MERS-CoV) vaccines. The S protein of MERS-CoV has been identified as the most promising immunogen [65], which has been identified as a balanced Th1 /Th2 inducer and was evaluated in different vaccination regimens [41,65-71]. Hence, the prime aim of developing adenovirus-based MERS-CoV vaccines is to identify and validate the most promising vaccination route and vaccination regimen to provoke balanced immunogenicity responses. Regarding respiratory viruses, mucosal immunity is crucial in eliminating the pathogen at the infection site [72]. Both mucosal and systemic immunological responses are important for better protection [66]. IN vaccination elicits a strong, safe [72,73], and sustained immunological response even with single administration [41] over subcutaneous administration. Considering the induction of systemic immunity, the IM route was better than the intragastric route in single-dose administration, and the Ad41 vector was a better substitute for Ad5, but the Ad41-induced responses were unsustainable [66]. In addition to adhering to one administration route, a prime-boost regimen with different administration routes also induces promising immune responses [74]. The S protein in ChAdOX1 is equally effective in generating nAb responses in IN and IM routes [71]. When immunized with Ad5, weak nAb responses against the Ad5 vector can be compensated for via heterologous prime-boost vaccination, but Ad5 mounts good Th1 responses against MERS-CoV. The nAb titer and the Th1 response are important for patient survival from a MERS-CoV infection because CD8+ causes faster virus clearance and shorter exposure to the virus [68]. Unlike the SARS-CoV S protein [75,76], Ad5-MERS-S induced balanced Th1 and Th2 responses [65] and nAb responses. Therefore, it is important to know whether similar responses are generated if dromedary camels vaccinated with these candidate vaccines or patients convalescing from a MERS-CoV infection show the same antigenic response [67]. Genetic modification, like the incorporation of human tissue plasminogen activator (tPA) [69] and molecular adjuvants (cytoplasmic domain 40 ligand [CD40L]) [77], enhances immunogenicity and efficacy. Recent studies have focused on new adenovirus vectors [41,69,70,78-82], which are less exposed to humans to circumvent vector immunity.

Table 3 summarizes the progress of research on adenovirus-based SARS-CoV-2 vaccines. Adenovirus-based SARS-CoV-2 vaccines have been used since the COVID-19 pandemic to cease and control the spread of infection. Nevertheless, researchers are attempting to boost the overall immunogenicity response and the durability of the immune responses through various vaccination regimens, such as altering the vaccination dose and optimizing the booster regimens [83]. A single vaccination dose has been proven to be efficacious [84-88]. Still, booster regimens are being optimized: fractionating the prime dose [83] and allowing homologous or heterologous boostering [89,90]. Modifying the glycoprotein by tPA enhances protein expression [84] and immunogenicity. The prefusion-stabilized state of the glycoprotein produces robust protection against the clinical disease [91] and is being evaluated in clinical trials [92] and induces ACE2 interference [93]. Moreover, self-bio mineralization with CaP exterior to the S protein provides thermostability by making an excellent mineral shell [42,94]. An antigen combination (S and N) with dual-route priming [95] and truncated S1 [96] also provides better immune responses than wild-type S protein alone. Despite this, the current COVID-19 vaccines use the wild-type S protein, decreasing their efficacy against variants but still offering protection in severe and critical infections [97]. S1 and N as fusion proteins showed a robust neutralizing response, and it was sustained against the Beta and Gamma SARS-CoV-2 variants [98]. The Omicron BA.1 S carrier induced nAbs against Omicron BA.2 [99].

In the commercial adenovirus-based SARS-CoV-2 vaccine productions, Sputnik V is a combination of 2 adenoviruses (hAd26 and hAd5) that has higher efficacy than the other commercial adenovirus-based SARS-CoV-2 vaccines {Vaxzevri (ChAdOx-1), (Ad26.COV2.S), Convidecia (Ad5-nCoV)} [97,100]. EUA-approved IM vaccines are currently used to control infection, and there are no approved oral vaccines for SARS-CoV-2 [101]. In the experimental setup, IN [102] and oral vaccines produced protective immune responses, and the immunity elicited from oral vaccination is on par with the IN vaccination [101].

In summary, different aspects have been considered in producing adenovirus-vectored human CoV vaccines. For example, the full-length S protein SARS-CoV induces Th2-skewed immunogenic responses that weaken cellular immunity. In this regard, glycoprotein modification (S1 subunit, RBD, RBD, and RBD-Fc) has been used to maximize immunogenicity. In adenovirus-based MERS-CoV vaccinations, almost all vaccine candidates have used the S antigen because it contains the RBD, the entry receptor of the viruses. Most importantly, nAbs are directed against the RBD, which is a conserved and highly antigenic region. Moreover, the S antigen of MERS-CoV is a balanced Th1/Th2 inducer. Therefore, the adenovirus-based MERS-CoV vaccinations are highly focused on vaccination platforms and regimens instead of antigen selection. In addition, adenovirus-based SARS-CoV-2 vaccines have also been assessed to upgrade the overall immune response and the durability of the immune response.

Table 4 lists the progress of research on Ad-based animal CoVs. The Ad5 vector-based vaccines expressing the S protein [103,104] or core neutralizing epitope (COE) protein [105] of the porcine epidemic diarrhea virus (PEDV) have been shown to elicit efficient humoral immune responses, significant viral shedding in pigs, and passive lactogenic immunity for newborn piglets. Most importantly, the single vaccination regimen provides adequate, efficient, and complete protection in farrowing sows, highlighting the important utility of the vaccine in reducing vaccination stress during pregnancy [103]. In addition, they have cross-neutralizing titers against different strains of PEDV [103,105]. The Ad5 vectored full-length S protein of the porcine respiratory coronavirus (PRCV) harbored neutralizing antibodies [106]. Transmissible gastroenteritis virus (TGEV) and PRCV show complete 2-way cross-neutralization [107]. Hence, the Ad5 vector expressing the common neutralizing epitopes might provide an attractive model for inducing immunity at the mucosal and enteric surfaces [108]. The S protein of feline CoVs (FCoVs) (especially the feline infectious peritonitis virus, the cause of feline infectious peritonitis) has been associated with the risk of antibody-dependent enhancement [109], where antibodies generated by the vaccine may worsen disease upon infection. Therefore, the Ad5 vector expressing the N protein of FCoV induced cellular immunity responses, effectively resisting a viral invasion [110]. Combining the S and M proteins of bovine coronavirus [111] in the Ad5 vector elicited effective humoral immune responses. This suggests advances in the combined vaccines in the veterinary and human fields. The abortive replication nature of Ad vaccines in the swine small intestine during the Ad-based S protein of TGEV [112] vaccination and the route dependency of humoral immune responses during Ad5-based TGEV and infectious bronchitis virus [113,114] vaccines reveal the constraints of the Ad-based swine and poultry coronavirus vaccine production.

CoV vaccines used in animals rely on traditional platforms, such as live and recombinant vaccines. Nevertheless, with the significant advances in vaccine technology and the critical role of CoVs in public health, it is essential to develop vaccines using more diverse platforms. The SARS-CoV-2 pandemic has highlighted adenovirus vectors as potent vaccine candidates. Adenovirus vectors are simple to engineer, cost-effective, and quick to manufacture. In addition, they are relatively safe and immunogenic in humans and do not require cold chain storage. These attributes make them well-suited for equitable global distribution and veterinary applications. Therefore, adenovirus vectors have significant potential as an effective vaccine platform for addressing CoV infections in humans and animals.