A China-developed adenovirus vector-based COVID-19 vaccine: review of the development and application of Ad5-nCov

ABSTRACT

Introduction

The global spread of COVID-19 has prompted the development of vaccines. A recombinant adenovirus type-5 vectored COVID-19 vaccine (Ad5-nCoV) developed by Chinese scientists has been authorized for use as a prime and booster dose in China and several other countries.

Areas covered

We searched published articles as of 4 May 2023, on PubMed using keywords related to Adenovirus vector, vaccine, and SARS-CoV-2. We reported the progress and outcomes of Ad5-nCov, including vaccine efficacy, safety, immunogenicity based on pre-clinical trials, clinical trials, and real-world studies for primary and booster doses.

Expert opinion

Ad5-nCoV is a significant advancement in Chinese vaccine development technology. Evidence from clinical trials and real-world studies has demonstrated well-tolerated, highly immunogenic, and efficacy of Ad5-nCoV in preventing severe/critical COVID-19. Aerosolized Ad5-nCoV, given via a novel route, could elicit mucosal immunity and improve the vaccine efficacy, enhance the production capacity and availability, and reduce the potential negative impact of preexisting antibodies. However, additional research is necessary to evaluate the long-term safety and immunogenicity of Ad5-nCoV, its efficacy against emerging variants, its effectiveness in a real-world context of hybrid immunity, and its cost-effectiveness, particularly with respect to aerosolized Ad5-nCoV.

KEYWORDS:

• adenovirus type-5
• COVID-19 vaccine
• Ad5-nCov
• aerosol inhalation
• safety
• effectiveness
• prime-boost strategy
• immunogenicity

1. Introduction

Vaccines are a successful and cost-effective method against viral diseases, including COVID-19 [1]. Novel vaccine technologies, like nucleic acid vaccines and viral vector vaccines, offer several advantages over traditional vaccine platforms: Novel technologies like nucleic acid and viral vector vaccines offer advantages over traditional platforms: faster development, modification for new variants, and safer than live attenuated vaccines. In addition, these technologies can avoid biosafety risks associated with the production of certain pathogens required for traditional vaccines [2–4]. Furthermore, viral vector vaccines are relatively inexpensive and more convenient to be shipped and stored, as compared to mRNA vaccines [5,6].

Various viral vectors, including adenovirus (Ad), modified vaccinia ankara (MVA), varicella-zoster virus (VZV), vaccinia virus, measles virus, rhabdovirus, and live attenuated influenza vaccine virus, have been developed to combat human immunodeficiency virus (HIV), tuberculosis, respiratory syncytial virus (RSV), Malaria, Ebola, and COVID-19 [7–18]. Adenovirus is widely used due to its broad target cell range, large packaging capacity, minimal risk of insertional mutagenesis, early innate immunity, high titer induction, well-studied genome and replication machinery, and established manufacturing processes [3,19]. Both human Ad and nonhuman Ad strains, such as chimpanzee adenovirus, gorilla adenovirus, rhesus adenovirus, have been studied extensively for the development of a COVID-19 vaccine [17,20–24].

There are four replication-deficient Ad vector-based COVID-19 vaccines have been approved for immunization campaign worldwide as of 22 November 2022, including Ad5-nCoV from CanSino, Ad26.COV2.S from Janssen/Johnson & Johnson, Sputnik V using Ad26 and Ad 5 from Gamaleya Research Institute, and the ChAdOx1 nCoV-19 vaccine using chimpanzee Ads from Oxford-AstraZeneca [25–29]. Each of the four Ad-Spike vaccines was shown to be safe and effective in reducing the severity of COVID-19, hospitalizations, and fatalities associated with it. Ad5-nCoV is the only viral-vector COVID-19 vaccine developed in China, as well as the first candidate vaccine to be studied in humans [30]. It can be administered via intramuscular (IM) injection or aerosolized inhalation. figure 1 illustrates the key milestones for the development of the Ad5-nCoV, as well as researches conducted to improve vaccination strategies [25,31–39].

Figure 1. Development timeline of Ad5-nCov.

Note: Ad5-nCoV: a recombinant adenovirus type-5 vector-based COVID-19 vaccine from CanSino; IM: intramuscular injection; NMPA: National Medical Products Administration; EUL: Emergency Use Listing

To gather information for this vaccine profile, we searched published articles as of 4 May 2023 through ‘PubMed,’ using ‘adenovirus vector’ and ‘vaccine’ and ‘SARS-CoV-2’ with no other restrictions, and then screen abstract of selected papers one-by-one to finally identify and include reports that focused on the development and application of Ad5-nCov, including vaccine efficacy, safety, immunogenicity based on pre-clinical trials, clinical trials and real-world studies for primary and booster doses.

2. Pre-clinical studies

2.1. Vaccine design

The development of Ad5 vectored COVID-19 vaccine involves various strategies, such as recombinant or hybrid Ad5 expressing full-length genome or receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, or nucleocapsid protein of the SARS-CoV-2, or dual antigen (SARS-CoV-2 spike and nucleocapsid proteins) [40–44]. To design Ad5-nCoV, Chinese scientist Chen Wei and her team drew from their successful experience in developing an Ad5-vectored Ebola vaccine that they utilized E1/E3 deleted replication-defective Ad5 that encodes the full-length, mammalian-expression-optimized Spike gene with a tissue plasminogen activator (tPA) signal peptide [17]. The structure of the adenovirus genome and vector is illustrated in figure 2.

Figure 2. Adenovirus genome and vector structure. A) Adenovirus particlestructure comprises an icosahedral shape without an envelope, and iscomposed of hexon protein, penton proteins, and fibrin. B) Adenovirusgenome structure is around 36kb in total length, containing 4 earlygenes (E1~E4) and 5 late genes (L1~L5), with each end possessing aninverted terminal repeat (ITR). C) The first-generation adenovirusvector is roughly 35kb in length. The vector is replication-deficientas the E1 gene associated with adenovirus replication is deleted.Additionally, the E3 gene, which is unrelated to replication, is alsodeleted, thus allowing the vector to carry the exogenous target geneup to approximately 8kb.

2.2. Vaccination via different routes

In addition to the traditional IM injection of viral-vector COVID-19 vaccines, mucosal routes, such as intranasal (IN) or oral immunization were extensively studied [45–49]. By eliciting mucosal immunity, respiratory pathogens can be captured and neutralized on the surfaces of the respiratory tract, which could in turn reduce the transmission of SARS-CoV-2 and the severity of the disease.

Ad5-nCoV induced robust immune response in wild-type BALB/c mice via IM and IN administration of a single dose, including robust Spike-specific IgG, anti-SARS-CoV-2-specific neutralizing immunity, as well as IFN-γ, TNF-α and IL-2-producing CD8+ T cells or CD4+ T cells, while Spike-specific IgA was only observed to be induced through IN administration [50]. A single dose of Ad5-nCoV via IM and IN routes provided complete protection against upper respiratory tract and lung infections in mice and significantly reduced viral replication in ferret’s upper respiratory tract.

In non-human primate (NHP) studies of rhesus macaques, nebulization inhalation was suggested as the optimal route for Ad5-nCoV mucosal administration, as it induced higher and longer-lasting serum Spike-specific IgG and S-RBD-specific IgA titers than IN and intratracheal aerosol administration [51].

2.3. Heterologous prime-boost schedule

Currently, available viral-vector COVID-19 vaccines have been extensively studied in animal models using heterologous prime-boost techniques, as demonstrated in various research studies [52–56]. Heterologous prime-boost strategies, such as combination of Ad5-nCoV as primary or booster immunization with other COVID-19 vaccines developed in China, including inactivated, recombinant subunit, or mRNA vaccine, have been shown to enhance Nabs and Th1 biased T cell responses in a mouse model, thereby providing broader protective immunity. Furthermore, the use of Ad5-nCoV as the primary immunization in a prime-boost regimen was found to induce higher levels of Nabs and promote the immune response regulation [54].

3. Application of Ad5-nCov in human

3.1. The profile of safety and acceptability

3.1.1. Safety profile in clinical trials

Ad5-nCoV given via both IM injection and the aerosol inhalation (see figure 3) in clinical trials showed high tolerability and short- and long-term safety profile [25,31–33,57,58]. Most adverse reactions (AR) occurred within 28 days post-vaccination, were temporary, and ranged in severity from mild to moderate, with the majority resolving in as little as three days. The most common ARs were injection-site pain (occurring in over half of participants in different studies with IM administration), respiratory-related reactions (such as dry mouth and pharyngeal swelling, occurring in 4%-9% of participants via aerosol inhalation), and systemic reactions including fever (27%-48%), fatigue (13%-44%), and headache (15%-39%), which were consistent for both vaccination routes. Severe ARs were mainly grade 3 fever that resolved within four days without medication.

Figure 3. Schematicsof aerosolized Ad5-nCoV inhalation. The recombinant adenovirustype-5 vectored COVID-19 vaccine (Ad5-nCoV) utilizes a non-pathogenicadenovirus vector to deliver the genetic material of the S protein,resulting in precise immune system activation without the risk ofinfection. Additionally, when administered via oral inhalation usinga nebulizer, the vaccine can be aerosolized into particles anddelivered to the lungs, leading to the stimulation of a triple immuneresponse involving humoral, cell, and mucosal immunity.

3.1.2. Factors influence the incidence rate of AR

In comparison to IM Ad5-nCoV and inactivated COVID-19 vaccines, participants who received an aerosolize Ad5-nCoV reported a lower or similar AR incidence [36]. High preexisting Ad5 immunity (titer of > 1:200 vs ≤ 1:200) was associated with significantly fewer occurrences of any or grade 3 fever who received IM injection, but this association was not observed in individuals who received aerosolized Ad5-nCoV [25,31,33,36]. In addition, a phase 2b clinical research found that IM Ad5-nCoV showed lower reactogenicity in participants aged 60 years and older than those aged 6–17 years and 18–59 years [57].

3.1.3. Safety profile of prime-boost regimen

Heterologous prime-boost regimens of Ad5-nCoV were tolerable and consistent with single dose safety profile, with no vaccine-associated SAEs reported up to 12 months following immunization. All cellular responses elicited by primary or booster doses of Ad5-nCoV were TH1-skewed, reducing the risk of vaccine-enhanced disease.

After the first vaccination, adverse events (AE) were lower for homologous booster dose of IM Ad5-nCoV injection, consistent with other adenovirus-vectored COVID-19 vaccines [58]. In contrast, aerosolized Ad5-nCoV vaccination had more frequent and severe adverse events after the booster, similar to mRNA-based and recombinant protein COVID-19 vaccines [59–61]. Heterologous prime-boost regimens of Ad5-nCoV were tolerable and consistent with single dose safety profile, with no vaccine-associated serious adverse events (SAEs) reported up to 12 months following immunization [62]. All cellular responses elicited by primary or booster doses of Ad5-nCoV were TH1-skewed, reducing the risk of vaccine-enhanced disease.

3.1.4. Rare adverse events post-authorization

Real-world evidence showed that common transit adverse reactions to adenoviral vector-based COVID-19 vaccines were similar to clinical trials, but some SAEs were rarely reported to be associated with Ad26.COV2.S and/or ChAdOx1 nCoV-19 vaccines, but not with Ad5-nCoV, such as anaphylaxis, thrombotic thrombocytopenia syndrome (TTS), Guillain‐Barré syndrome (GBS), IgA vasculitis, bleeding episodes, and Acute Transverse Myelitis (ATM) [63–71].

An analysis of passive epidemiological surveillance data in Mexico revealed that the unadjusted incidence of GBS following Ad5-nCoV administration was 1.68/1,000,000 doses, which was lower than the incidence observed in the recipients of Ad26.COV2-S (3.86/1,000,000 doses) and BNT162b2 (1.92/1,000,000 doses) [72]. The unadjusted incidence of psychological/psychiatric symptoms following Ad5-nCoV administration was 2.44 cases per 100,000 doses, and the stroke was 1.17 cases per 1,000,000 doses [73,74]. However, no causal association between Ad5-nCoV and GBS, psychological/psychiatric symptoms, or stroke has been established. Furthermore, a single dose of Ad5-nCoV was safe for patients with benign childhood epilepsy with centrotemporal spikes and orthotopic liver transplant recipients [75,76].

3.2. Immunogenicity

Both IM and aerosolized Ad5-nCoV could induce antibody and T-helper-1 cell-bias T-cell responses [25,31–33,51].

3.2.1. Humoral immunity elicited by IM vaccination

Evidence from several clinical trials in healthy individuals aged 6 years and older showed that a single dose of could induce a significant immune response [25,31,57], with GMTs of ELISA anti-RBD antibodies to be 169.8 to 1091.6, GMTs of neutralizing antibodies (Nabs) to pseudovirus to be 35.8 to 168.0, and a seroconversion rate of Nabs to be 75.9% [32]. Among those, GMTs of Nabs responses to live SARS-CoV-2 were observed to decline with increasing age be 16.2 to 18.3 in adults aged 18–59 years [31], and it was found that children and adolescents had a stronger immune response than adults and older people [57]. The T-cell responses were found in 74%–84% of the patients after immunization with a single dose of intramuscularly given Ad5-nCoV [31].

3.2.2. Humoral immunity elicited by aerosol vaccination

Two doses of aerosolized Ad5-nCoV elicit3e comparable Nab responses against live virus (107 for 0.2 mL of 1 × 10¹⁰ viral particles per mL and 105 for 0.1 mL of 1 × 10¹⁰ viral particles per mL) to one dose of IM injection (95 for 1.0 × 10^{1 1} viral particles per mL) [33]. An aerosolized booster dose given at 28 days after a single IM injection resulted in stronger ELISA anti-RBD IgG (2013 EU/mL at 28 days post-vaccination) and Nab responses (396 EU/mL), than aerosol or IM vaccination alone. An aerosol vaccination with only one-fifth of the IM dose triggered similar IFN-γ response to that of the IM injections. Moreover, inhalation of aerosolized Ad5-nCoV induced a greater ratio of Nabs to total antibodies and robust mucosal immunity compared to IM vaccination.

3.2.3. T-cell response

After the first dose of IM or aerosolized Ad5-nCoV vaccine, RBD-specific antibodies and Nabs significantly increased at day 14 and peaked at day 28 [31]. Meanwhile, the T cell response peaked earlier at day 14, indicating T cells might provide earlier protection against symptomatic COVID-19 after single-dose vaccination [33]. During a mouse model pre-clinical trial, it was found that the type I interferon pathway could be a crucial factor in generating optimal antiviral T cell responses after SARS-CoV-2 infection [77]. Both aerosolized Ad5-nCoV and IM Ad5-nCoV studies have demonstrated that CD4 T cells primarily secrete T-helper-1 cell cytokines, including IFN-γ and IL-2, rather than T-helper-2 cell cytokines like IL-4 and IL-13, which is in line with other adenovirus-vectored or mRNA-based COVID-19 vaccines research [78,79].

3.2.4. Impact of preexisting Ad5 neutralizing antibody

Preexisting anti-Ad antibody may be an unavoidable issue for ad-vector vaccines, particularly for most studied Ad5. This issue may have a negative impact on the clinical translation of Ad5 vectors and, as a result, may lead to a reduction in the immune response that is elicited by Ad5 vectored vaccines [19]. A high preexisting Ad5 neutralizing antibody titer (>200 vs ≤ 200) was observed to compromise the SARS-CoV-2 neutralizing antibody titers in participants who received aerosol or IM vaccination [25,31,33,36], and significantly reduce the SARS-CoV-2 spike protein-specific IFN-γ response at day 14 after initial and booster dose of aerosolized vaccination [33]. Additionally, a limited 1.7-fold increase of Nab titers 28 days after the second dose of a homologous prime-boost regimen of Ad5‐nCoV with a 56-day interval was found in individuals aged 6 and above [57].

3.3. Efficacy and effectiveness

As aerosolized Ad5-nCoV was not licensed for using as booster dose in China until late 2022, data referring to efficacy and real-world effectiveness gathered in this review was mainly about IM administered Ad5-nCoV.

3.3.1. Efficacy in clinical study

A single dose of Ad5-nCoV was shown to be effective in individuals aged 18 and older in a phase 3 trial that was conducted across many centers and included participants from a geographically and ethnically varied population [32]. Based on more than 10 000 recipients in each group, the vaccine efficacy 28 days postvaccination was 57.5% against symptomatic COVID-19 and 91.7% against severe disease, which is comparable to other single-dose or two-dose scheduled adenoviral-vectored vaccines. The corresponding 14 days post-vaccination efficacies were found to be 63.7% and 96.0%, respectively.

3.3.2. Real-world evidence for vaccine effectiveness (VE)

Observational studies conducted in China and Mexico during the Delta and Omicron waves reported high VE rates of over 70% against severe/critical COVID-19 and even 100% against death, comparable to other vector-based COVID-19 vaccines [80–82]. However, the VE of Ad5-nCoV against symptomatic COVID-19 and SARS-CoV-2 infection may be diminished and varies among different studies, particularly against emerging variants.

A cohort study conducted in China indicated that a single dose of Ad5-nCoV provide moderate two-month postvaccination VE of 61.5% and 67.9% against symptomatic COVID-19 and pneumonia during the Delta (B.1.617.2) outbreak, while another 6-month follow-up cohort study in Mexico revealed a much lower adjusted VE of only 18% against laboratory-confirmed COVID-19 illness during Delta predominance (2.9-fold lower to that observed before Delta predominance) [83,84]. On the other hand, a case-control study in Anhui province reported an adjusted VE of 65.8% (95% CI: 12.8–86.6%) against Omicron BA.2.2 infection, while another matched case-control study in Shanghai reported a real-world VE of only 13.2% against SARS-CoV-2 Omicron BA.2 infection [85,86]. A retrospective cohort study in Mexico found a symptomatic breakthrough infectious rate of 4.1% (224/5360) in Ad5-nCoV recipients, which might indirectly prove a moderate of Ad5-nCoV against symptomatic COVID-19 [87].

The variability in results among different studies can be attributed to various factors, including differences in study design, limitations in the strength of evidence from observational studies, varying intervals between vaccination and outcomes, sample sizes, and others. In addition, several studies have observed a decline in VE over time after vaccination, which raises the possibility of the need for a booster dose [84,88]. Furthermore, one study found that Ad5-nCoV had VEs of 39.3% against Omicron BA.2 variant infection 3–12 weeks after the first dose, but only 16.0% at 13–24 weeks after the homologous booster dose, suggesting that a heterologous prime-boost regimen may be better than a homologous schedule [86].

3.4. Combination of Ad5-nCov with other COVID-19 vaccines

The emergence of SARS-CoV-2 variants, particularly the Omicron variant, poses significant challenges to the development of vaccines and the immunization strategies for existing COVID-19 vaccines, due to their ability to evade immunity [89–92]. Various studies have focused on booster strategies for currently used COVID-19 vaccines in response to waning humoral immunity, emerging variants, observed severe life-threatening ARs following some COVID-19 vaccines and so on [93,94]. To date, heterologous prime-boost vaccine regimens for SARS-CoV-2 have been demonstrated to produce higher, broader, and more long-lasting protective immunity while maintaining a similar safety profile [35,36,62,95–97].

3.4.1. Ad5‐nCov as first and second booster dose following inactivated COVID-19 vaccine

Numerous investigations on IM and aerosolized Ad5‐nCoV as a first or second heterologous booster dose following two-dose or three-dose inactivated COVID-19 vaccine were conducted [36,58,96,98]. A first booster shot of Ad5-nCoV vaccine via both IM injection and aerosolize inhalation could generate a stronger and more persistent (lasting at least 12 months) immune response in comparison to a three-dose inactivated COVID-19 vaccine schedule, while also displaying significant cross-neutralization activity against the Delta variant [36,62,96]. In a cohort study, a booster dose of Ad5­nCOV was shown to elicit equivalent anti-Omicron-RBD antibody levels to those elicited by Omicron variant breakthrough infection following two doses of inactivated vaccine [99].

Aerosolized Ad5‐nCoV, in particular, could induce stronger Nabs responses and RBD specific IgA response compared to IM injection of Ad5‐nCoV, and even stronger than those induced by other reported heterologous booster regimens combing COVID­19 vaccines of different platforms, including mRNA, viral-vector, and protein-adjuvant vaccines (as listed in Table 1) [35,36,62,100,101]. Additionally, aerosolized Ad5‐nCoV might effectively induce a higher level of Nabs against Omicron variants than a homologous third dose of inactivated COVID-19 vaccine and heterologous regimen with RBD subunit vaccine following a two-dose of inactivated COVID-19 vaccine [95].

Table 1. Listing of studies addressing heterologous prime-boost techniques of COVID-19 vaccines based on different platforms.

Author	Immunization interval	Reference number	Arms	Age of participants	Sample size	The peak of neutralizing antibodies against the wild-type SARS-CoV-2 (IU/mL)
Li, J.X., et al.	3–9 months	Ref [36].	Low dose group: low dose (1.0 × 10^1 1 viral particles per mL; 0.1 mL) aerosolized Ad5-nCoV as third dose following two-dose of CoronaVac; High dose group: high dose (1.0 × 10^1 1 viral particles per mL; 0.2 mL) aerosolized Ad5-nCoV as third dose following two-dose of CoronaVac; CoronaVac group: three-dose of CoronaVac	≥18 years	140 per group	Two-dose of CoronaVac + low dose aerosolized Ad5-nCoV: 6054.1 Two-dose of CoronaVac + High dose aerosolized Ad5-nCoV: 4221.3 Two-dose of CoronaVac + CoronaVac: 245.3
Li J, et al.	3–6 months	Ref [93].	Heterologous prime-booster group: one-dose or two-dose of CoronaVac + intramuscular Ad5-nCoV Homologous booster dose group: one-dose or two-dose of CoronaVac + CoronaVac	≥18 years	Primed with one-dose CoronaVac: 50 per group; Primed with two-dose CoronaVac:100 per group	Two-dose of CoronaVac + Ad5-nCoV: 616.9 Two-dose of CoronaVac + CoronaVac: 105.0 One-dose of CoronaVac + Ad5-nCoV: 170.0 One-dose of CoronaVac + CoronaVac: 40.0
Jin P, et al.	28 days, 56 days, 5 months, 6 months	Ref [35].	CV/ZF/ZF (D0-D28-M5): prime dose of Ad5-nCoV (CV), booster doses of ZF2001 (ZF) at day 28 and month 5; CV/ZF (D0-M5): prime dose of Ad5-nCoV, booster dose of ZF2001 at month 5; CV/ZF/ZF (D0-D56-M6): prime dose of Ad5-nCoV, booster doses of ZF2001 at day 56 and month 6; CV/ZF (D0-M6): prime dose of Ad5-nCoV, booster dose of ZF2001 at month 6	≥18 years	A total of 120	CV/ZF/ZF (D0-D28-M5) regimen: 335.0 CV/ZF/ZF (D0-D56-M6) regimen: 441.3 CV/ZF (D0-M5) regimen: 282.8 CV/ZF (D0-M6) regimen: 294.1
Munro APS, et al.	>84 day		BNT/BNT+BNT	>30 years	96	BNT/BNT+BNT: 955.7
			BNT/BNT+m1273		92	BNT/BNT+m1273: 1078.5
			BNT/BNT+ChAd		98	BNT/BNT+ChAd: 507.5
			BNT/BNT+NVX		103	BNT/BNT+NVX: 409.2
		Ref [97].	BNT/BNT+VLA		99	BNT/BNT+VAL: 154.4
			BNT/BNT+Ad26		89	BNT/BNT+Ad26: 769.8
			BNT/BNT+CVn		94	BNT/BNT+CVn: 260.2
	>70 day		ChAd/ChAd+ChAd		100	ChAd/ChAd+ChAd: 103.1
			ChAd/ChAd+NVX		96	ChAd/ChAd+NVX: 388.4
			ChAd/ChAd+BNT		95	ChAd/ChAd+BNT: 865.9
			ChAd/ChAd+VLA		95	ChAd/ChAd+VLA: 107.9
			ChAd/ChAd+m1273		98	ChAd/ChAd+m1273: 1265.0
			ChAd/ChAd+Ad26		101	ChAd/ChAd+Ad26: 300.7
			ChAd/ChAd+CVn		105	ChAd/ChAd+CVn: 199.3

IU: international unit; Ad5-nCoV (CV): a recombinant adenovirus type-5 vector-based COVID-19 vaccine from CanSino; ZF2001 (ZF): a recombinant protein COVID-19 vaccine from ZFSW; BNT: BNT162b2, an mRNA COVID-19 vaccine from Pfizer; m1273: mRNA-1273, a mRNA-based COVID-19 vaccine from Moderna; ChAd: ChAdOx-nCov19, a chimpanzee Ads vectored COVID-19 vaccine from AstraZeneca; NVX: NVX-CoV2373, a recombinant protein COVID-19 vaccine from Novavax; VLA: VLA2001, an inactivated COVID-19 vaccine from Valneva; Ad26: Ad26.COV2.S, replication-deficient adenovirus vector COVID-19 vaccine from Janssen; CVn: CVnCoV, a mRNA-based COVID-19 vaccine from Curevac.

Furthermore, a fourth dose (second booster) of Ad5-nCoV (aerosolized or IM) after receiving three doses (prime two-dose and first booster dose) of inactivated COVID-19 vaccine could result in significantly higher serum Nab GMT than four doses of inactivated COVID-19 vaccine (672.4 in aerosolized Ad5-nCoV group, 582.6 in IM Ad5-nCoV group vs 58.5 in inactivated COVID-19 vaccine group) [98].

3.4.2. Ad5‐nCov following other COVID-19 vaccines

In Argentina, an extensive cohort study was carried out to examine 15 different two-dose schedules, both homologous and heterologous, in individuals who had been primed by either Sputnik V C1 (rAd 26), ChAdOx1-S, or BBIBP-CorV [102]. The study also included two heterologous prime-boost regimens in which Ad5-nCoV was used as a booster dose following one dose of either Sputnik V C1 or ChAdOx1-S. The results showed that a heterologous booster dose of Ad5-nCoV elicited a stronger humoral immunity than corresponding homologous regimens and heterologous regimen using BBIBP-CorV as a booster. Moreover, the immune response induced by the Ad5-nCoV booster was comparable to that produced by heterologous regimens using other viral-vector vaccines.

3.4.3. Ad5‐nCov used as prime dose

Research has also been done on various prime-boost regimens using Ad5-nCoV as the prime dose. In a phase 4 clinical trial conducted in China, a combination of Ad5-nCoV and a protein-subunit-based COVID-19 vaccine induced robust humoral immune responses in adults, with no increase in specific T-cell response, particularly with a 5 to 6 months immunization interval [35]. Two real-world small-scale cohort studies carried out in Mexico suggested that primed with Ad5-nCoV and then subsequently given booster doses of ChAdOx1-S-nCoV-19, Ad26.COV2.S, BNT162b2, or mRNA-127z at a 4–5 months interval showed stronger humoral immunity compared to those who did not receive any booster dose [103,104]. Another cohort study indicated that a single dose of IM Ad5-nCoV resulted in persistent Nabs for up to eight months, albeit with reduced levels against Omicron BA.1 and BA.5.1.6 and no detectable levels against BQ.1.3 and XBB.1. However, when a heterologous booster shot of mRNA-1273 was administered, the neutralizing antibody levels increased significantly, particularly in those who had previously been infected [105].

4. Expert opinion

Ad5-nCoV is a significant milestone in Chinese vaccine development technology, presenting the first COVID-19 vaccine developed and tested in human in China, and the first Chinese-developed viral-vector COVID-19 vaccine to receive the WHO Emergency Use Listing. Evidence from phase 1 to phase 3 clinical trials proved that a single dose of intramuscularly administered Ad5-nCoV is well-tolerated, highly immunogenic, and effective in preventing severe/critical COVID-19, supporting its approval for emergency use in several countries since early 2021.

To enhance the efficacy of viral-vector COVID-19 vaccines, aerosolized Ad5-nCoV was investigated as a means to induce mucosal immunity, given that SARS-CoV-2 primarily spreads through the respiratory tract. It was administered as a booster dose following either IM Ad5-nCoV or an inactivated vaccine, inducing higher levels of Nabs, cellular and mucosal immunity without the need for invasive vaccination. Using only one-fifth of the IM Ad5-nCoV dosage, aerosolized Ad5-nCoV significantly increased the production capacity of the vaccine. This convenient, needle-free vaccination route could not only reduce the injection-site adverse reactions but also improve the public willingness to be vaccinated while addressing the issue of handling used sharp tools.

The prime-boost strategy is an effective method to combat waning Nab levels and emerging SARS-CoV-2 variants. However, pre-existing anti-Ad antibodies, particularly with Ad5 that is highly prevalent, may compromise the efficacy of booster Ad vectored vaccines. To address this challenge, scientists recommend using a homologous prime-boost regimen with Ad5-nCoV, with a minimum interval of 6 months between sequential doses, or using a booster aerosolized Ad5-nCoV after a prime IM Ad5-nCoV. Alternatively, a heterologous prime-boost regimen could be used, such as using IM Ad5-nCoV as prime vaccination followed subunit protein vaccine or mRNA vaccine, or administering IM or aerosolized Ad5-nCoV as a booster dose following an inactivated or other viral-vector COVID-19 vaccines such as Sputnik V and ChAdOx1-S. This approach could help reducing the negative impact of preexisting Ad5 antibodies and supporting the flexible use of different platform-based COVID-19 vaccines to effectively respond to fluctuating vaccine supplies. However, further extensive studies are required to evaluate the immunization strategy of Ad5-nCoV in the context of hybrid immunity induced by both vaccination and infection worldwide, particularly for individuals who have already received a heterologous booster dose of Ad5-nCoV.

To ensure the long-term safety profile of Ad5-nCoV, it is essential to conduct rigorous post-authorization safety surveillance and observational studies and continually monitor any rare safety signals following Ad5-nCoV vaccination. Further studies are needed to evaluate the long-term immunity provided by different vaccination regimens, the effectiveness of Ad5-nCoV against emerging SARS-CoV-2 variants, the effectiveness of aerosolized Ad5-nCoV to block transmission, real-world effectiveness and cost-effectiveness of Ad5-nCoV. Additionally, the assay used to detect mucosal Spike-specific IgA, which has the potential to improve the efficacy of Ad5-nCoV against SARS-CoV-2 infection, still needs to be optimized. Finally, this platform could be potentially used for the development of vaccines against other difficult-to-control infectious pathogen such as tuberculosis and HIV.

Article highlights

• Ad5-nCoV is a remarkable achievement in Chinese vaccine innovation.

• Clinical trials showed that a single intramuscular dose of Ad5-nCoV was safe, immunogenic, and effective in preventing severe/critical COVID-19.

• Aerosolized Ad5-nCoV was explored as a way to induce mucosal immunity and enhance the efficacy of viral-vector COVID-19 vaccines.

• The production capacity of the vaccine was significantly increased by using only one-fifth of the intramuscular Ad5-nCoV dose for the aerosolized Ad5-nCoV.

• To overcome the negative impact of preexisting anti-Ad5 antibodies, a homologous prime-boost regimen with Ad5-nCoV given at least 6 months apart, or using aerosolized Ad5-nCoV as a booster dose, or a heterologous prime-boost regimen might be optimal strategies.

• Further studies are needed to evaluate the long-term safety and immunogenicity of Ad5-nCoV, the efficacy against emerging variants, effectiveness in a real-world context of hybrid immunity, as well as the cost-effectiveness of Ad5-nCoV, particularly with respect to aerosolized Ad5-nCoV.

• The Ad5-nCoV platform could be potentially used for developing vaccines against other challenging infectious pathogens.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or material discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or mending, or royalties.

Reviewer disclosures

A reviewer on this manuscript has disclosed that they have previously worked with the owner of the company that licensed the Ad5 manufacturing technology to Cansino Biotech. Peer reviewers on this manuscript have no other relevant financial or other relationships to disclose.

Authors contributions

Shen-Yu Wang contributed to drafting and revising of this manuscript. Wen-Qing Liu and Yu-Qing Li contributed to assistant drafting of this manuscript. Jing-Xin Li and Feng-Cai Zhu contributed to critically review the manuscript. All the authors have accessed and verified the data, and Jing-Xin Li and Feng-Cai Zhu are responsible for the decision to submit the manuscript.

Additional information

Funding

This study was supported by the National Natural Science Foundation of China (number 82173584 and 82222062), Jiangsu Provincial Science Fund for Distinguished Young Scholars (number BK20220064), and Jiangsu Provincial Key Project of Science and Technology Plan (number BE2021738).