Genetic-Based Vaccine Vectors

Cell Substrates for AdVV

Changes in regulatory requirements have been critical for advancing more efficient production methods, allowing for improved product development based on the adenoviral platform ( Fig. 68.4 ). The development of transformed cell lines for adenovector propagation, in contrast to the previous standard of avian leucosis-free primary CEFs, represented a major advance in viral vaccine production technology. These cell lines offer improved yields and stable production capacity. The development of these lines was exceptionally rigorous over many years in order to address important regulatory concerns regarding adventitious agents, tumorigenicity, and other safety and consistency-of-production issues.

Oversight and evaluation of the strengths and limitations of these cell substrates continue, based on guidelines created several years ago, ^, enabling improved characterization and availability of an increasing number of such lines. The PER.C6 TetR, HEK293, and GV11 cell lines, for example, have supported production of clinical-grade Ad5, Ad26, and simian adenoviruses, ChAd3, and ChAdOx1 for vaccines research in HIV-1, Ebolavirus, MERS-COV, Marburg virus, tuberculosis, malaria, and COVID-19 among others ( Fig. 68.4 ). ^,

AdVV as Vaccine Candidates: An Historical Perspective Shaped by Recent Epidemics

More than 51 human serotypes in seven subfamilies (A to G) of adenoviruses are known. ^, The adenovirus 5 serotype (Ad5) is derived from the adenoviral C subfamily and is the most common and well-characterized adenovirus serotype. However, the relatively high seroprevalence of Ad5 neutralizing antibodies in certain human populations ^{, ,} has suggested possible limitations to the global deployment of Ad5 vectors. Although pre-existing immunity has been shown to reduce the immunogenicity of Ad5 vaccines in mice, ^, rhesus monkeys, and potentially in humans, ^, it does not appear to completely block vaccine immunogenicity. Strategies to circumvent pre-existing immunity, such as engineering novel Ad5 vectors containing chimeric fiber and hexon virion proteins, chemically inhibiting antivector antibody binding with, for example, polyethylene glycol, or using oral–mucosal or nasal routes of administration, ^{, ,} are areas of active investigation. Another approach to circumvent pre-existing immunity is to use rare human Ad serotypes (e.g., Ad26, Ad35, Ad11, Ad4, Ad7) or adenoviral vectors derived from other species such sheep, pigs, cows, and nonhuman primates (NHP; e.g., ChAdOx1, GRAd32, ChAd36, ^, SAdV23 ⁹⁴ ). In recent years, numerous human- and simian-derived AdVV have been developed as vaccine candidates for HIV-1, ^, Ebola, RSV, Middle East Respiratory Syndrome (MERS), and COVID-19. ^,

Ad5 has been well characterized in a variety of animal models supporting its advancement in clinical studies. Ad5 vaccines and DNA-prime/Ad5-boost combinations conferred partial protection in rhesus macaques against multiple HIV isolates, including SHIV-89.6P, ^, SIVmac239, and SIVmac251. ^{, ,} Accordingly, Phase I and Phase II clinical studies with replication-defective adenoviral vectors for HIV-1 were advanced. The STEP study, a large Phase III efficacy study, evaluated the effect of an Ad5 vaccine encoding Gag , Pol , and Nef genes of HIV-1 on prevention of infection or controlling viral load. Although the Ad5 vaccine induced both humoral and limited cellular immunity, no reduction in HIV-1 acquisition or improvement in long-term control of postinfection viremia was observed. Further analyses revealed that persons with specific human leukocyte antigen (HLA) types, as well as those who developed a CD8+ T-cell response to certain Gag and Nef HIV-1 epitopes, selected against viruses that contained the vaccine epitope in vivo. Additionally, there was an unexpected increase in HIV-1 infection in a subgroup of vaccinees who were both uncircumcised and immune to Ad5 before vaccination during the first 18 months postimmunization. A follow-up prime-boost study using 505 DNA-prime rAd5-boost had a similar outcome and ended early due to futility. However, the Ad5 serology insights gained from these studies spurred research into additional adenovirus serotypes and nonhuman adenoviral vector approaches.

Seroepidemiology analyses of Ad serotypes suggest that pre-existing immunity to Ad11, Ad35, and Ad26 is less common than observed with Ad5, thereby making these vectors attractive candidates for vaccine development. ^, A prime-boost regimen of Ad26 and Ad35 vaccine vectors expressing the Ebola virus glycoprotein confers protection in NHP models, irrespective of prior Ad5 immunity. Ad26.ZEBOV is an Ad26 AdVV expressing the Ebola virus glycoprotein that was developed by Janssen as part of the public health response to the 2014–2016 West African Ebola epidemic. Ad26.ZEBOV is administered as a prime dose followed by an 8-week booster dose of MVA-BN-Filo, an MVA vaccine encoding glycoproteins from Ebola virus, Sudan virus, Marburg virus, and Tai Forest virus nucleoprotein. ^, Ad26.ZEBOV/MVA-BN-Filo became the first AdVV-vaccine regimen to receive regulatory approval from the European Medicines Agency (EMA) in July 2020 and is a prospective tool to mitigate future Ebola outbreaks. Ad26.ZEBOV priming induced antibody and T-cell responses to Ebola virus glycoprotein 14 days postimmunization during early safety and immunogenicity studies. Substantial strengthening of humoral and cellular immune responses following the MVA-BN-Filo booster was maintained for 8–12 months across multiple Phase I studies. The safety profile of Ad26.ZEBOV prime and MVA-BN-Filo boost support licensure in adults ^{, ,} and children. As conducting traditional randomized controlled efficacy studies of a prophylactic vaccine is only feasible with large outbreaks, manufacturers and regulators have relied on immunobridging analyses to infer the efficacy of Ad26.ZEBOV/MVA-BN-Filo against Ebola virus. Analyses using a fully lethal Ebola Kikwit NHP challenge model have estimated that antibody titers elicited by the prime-boost regimen should confer protection against mortality from Ebola virus. Janssen has also utilized the Ad26 vaccine platform to develop Ad26.RSV.preF, an AdVV expressing the prefusion spike protein of RSV. Ad26.RSV.preF induced Th1-skewed immunity and was protective against enhanced respiratory disease upon RSV challenge in murine models and during human challenge studies. Ad26.RSV.preF was 80% effective in protecting against lower respiratory tract illness in adults aged >65 years, supporting further clinical development.

In recent years, the utility of simian-derived adenoviruses as vaccine platforms for emerging infectious diseases (EID) has been demonstrated. Seroepidemiology studies have observed low levels of pre-existing neutralizing antibodies against chimpanzee adenoviral vectors in sub-Saharan Africa (1.7–18.7% for AdC6, AdC6, and AdC1 vs 55.8–95.8% for hAd5), the United States, and the United Kingdom. Simian-derived adenoviruses have shown promise as vaccine candidates for Ebola and MERS-CoV. A simian-derived replication-defective adenovirus (ChAd3) vaccine engineered to express the Ebola virus glycoprotein (ChAd3-EBO-Z) was immunogenic and induced protection against acute lethal challenges in rhesus macaques. When ChAd3-EBO-Z prime was boosted with a MVA vaccine also encoding the Ebola glycoprotein, more durable protection against lethal Ebola virus challenge was observed increasing from 5 weeks to 10 months. ChAd3-EBO-Z has been observed to be well tolerated and immunogenic in multiple clinical studies. Anti-Ebola virus glycoprotein antibody titers were detectable 1-month postimmunization, ^{, , , ,} were largely maintained through 12 months, and were higher in individuals who received MVA boosts. ^{, , , ,} ChAdOx1 is an AdVV vaccine platform developed by the University of Oxford that has been utilized to develop vaccines for EID. ChAdOx1 MERS is a simian-derived replication-defective adenovirus that expresses the spike glycoprotein of MERS-CoV. ^, Single-doses of ChAdOx1 MERS elicited neutralizing antibodies and cellular immune responses that conferred protection to humanized DPP4 mice and rhesus macaques from lethal MERS-CoV challenge, and reduced viral shedding and nasal discharge in Dromedary camels (i.e., the intermediate host of MERS-CoV). Single-dose ChAdOx1 MERS elicited humoral and cellular immune responses against MERS-CoV spike protein 14 days postimmunization and was well tolerated, and elucidation of the clinical efficacy of ChAdOx1 MERS is ongoing. The ChAdOx1 platform was used to develop AZD1222 (ChAdOx1 nCOV-19) to help mitigate the COVID-19 pandemic. Within the first year of deployment adenovirus-based COVID-19 vaccines derived from human adenoviruses (e.g., Ad26.COV.S, Ad5-nCoV, and Gam-COVID-Vac) and simian-derived adenoviruses (e.g., AZD1222) have demonstrated the utility of AdVV in combating an EID through their rapid deployment and development, as discussed next in context of the COVID-19 pandemic. ^{, , ,}

AdVV Platform Validation: Adenoviral Vectors as COVID-19 Vaccines

The rapid development of vaccines during the COVID-19 pandemic has yielded numerous adenovirus-vectored COVID-19 vaccine candidates. Four AdVV were among the first COVID-19 vaccines licensed for use, namely; AZD1222, Ad26.COV2.S, Ad5-nCoV, and Gam-COVID-Vac, a heterologous prime-boost combination of Ad26 and Ad5. Studies with other simian (e.g., GRAd32, ChAd36, ^, SAdV23 ⁹⁴ ) and human adenoviral vectors (e.g., human Ad49, single-cycle Ad6 ¹⁵⁴ ) are currently ongoing.

Characteristics of AdVV for COVID-19 Vaccines

The SARS-CoV-2 spike (S) glycoprotein is involved in angiotensin-converting enzyme (ACE) 2 receptor recognition, viral attachment, and entry into host cells. Functionally, the S glycoprotein is characterized by a short N-terminal signal peptide, an S1 domain that mediates host cell receptor binding via a receptor-binding domain (RBD), and an S2 domain that mediates virus membrane fusion. Due to its indispensable functions, first-wave adenovirus-based COVID-19 vaccine candidates were predominantly developed to target the S glycoprotein. ^, Other vaccine candidates such as AdCOVID specifically target the S glycoprotein RBD, while VXA-CoV2-1 ¹⁵⁸ and the hAd5 bivalent vaccine target both the SARS-CoV-2 nucleocapsid and S glycoprotein.

Once administered, adenovirus-vectored COVID-19 vaccines infect host cells and initiate production of the SARS-CoV-2 S glycoprotein (see Fig. 68.5 ). ^{, ,} AZD1222 and species E adenovectors enter host cells via the widely expressed coxsackie and adenovirus receptor, while Ad26 and other species D adenovectors utilize CD46 for host-cell entry. Currently licensed adenovirus-vectored COVID-19 vaccines are administered via a deltoid intramuscular (IM) injection ^{, , , ,} and other routes of administration are currently being explored, for example, intranasal and aerosolized. Preclinical evidence suggests that intranasal administration of AdVV can induce local respiratory tract mucosal immunity, reducing viral shedding, and conferring host protection from severe disease. ^{, ,} A Phase I study demonstrated that two doses of intranasally administered Ad5-nCoV elicited comparable humoral and cellular immune responses to a single IM immunization.

Current adenovirus-vectored COVID-19 vaccine candidates have been licensed as either single-dose or two-dose homologous prime-boost regimens. ^, Preclinical studies of AZD1222, ^{, ,} hAd5, and Ad26.COV2.S ^, demonstrated that although single-dose vaccination induces SARS-CoV-2 S glycoprotein-specific antibodies and cell-mediated immunity, a 4- or 8-week booster dose significantly increased titers of virus-neutralizing and RBD antibodies. Other adenovirus-based COVID-19 vaccines in development (e.g., Gam-COVID-Vac, Sad23LnCoV-S/Ad49L-nCoV-S ⁹⁴ ) utilize a heterologous prime-boost strategy with two different AdVV in order to minimize potential vaccine attenuation by a host immune response to AdVV.

Immunogenicity and Reactogenicity of Adenovirus-Based COVID-19 Vaccines

The humoral and cell-mediated immune response to adenovirus-based COVID-19 vaccines has been extensively characterized in exploratory analyses of large clinical trials. A single dose of AZD1222 induced B-cell activation and proliferation and production of S glycoprotein IgA, IgM, IgG1, and IgG3 antibodies. ^, Subsequent systems serology analyses demonstrated that anti-S glycoprotein neutralizing antibody titers and Fc-mediated functional antibody responses (namely, antibody-dependent neutrophil/monocyte phagocytosis, natural killer cell activation, and complement activation) were substantially enhanced by a second dose of vaccine. Polyfunctional IFN-γ+, TNF-α+, Th1-CD4+, and CD8+ T cells against the SARS-CoV-2 S glycoprotein peptides were induced 14 days following single-dose immunization ^, and were increased 28 days postsecond immunization. T-cell receptor β sequences revealed a substantial breadth and depth of S glycoprotein epitopes for AZD1222-induced CD4+ and CD8+ T-cell responses.

Immunogenicity analyses from Phase I and Phase II studies of single-dose Ad5-nCoV immunization demonstrated that neutralizing antibodies and IFN-γ+, TNF-α+, Th1-CD4+, and CD8+ T cells were induced within 14–28 days of vaccination. ^, Exploratory Phase I immunogenicity analyses with Ad26.COV2.S ^{, ,} illustrated that single-dose immunization elicited durable humoral responses, with a 1.8-fold reduction in neutralizing antibody titers, 8 months following immunization. S glycoprotein-specific IFN-γ+, CD4+, and CD8+ T-cell responses also showed durability and stability during this follow-up period. The immunogenicity of Gam-COVID-Vac was characterized in a Phase I/II study, wherein SARS-CoV-2 RBD-specific IgG antibody titers were observed 14 days following priming doses of either rAd26 or rAd5 and began to increase within 7 days of administering a booster dose of rAd5. S glycoprotein IFN-γ+, CD4+, and CD8+ T cells were induced following immunization, notably at 28 days following prime immunization and within 14 days of the rAd5 booster dose.

AZD1222, Ad26.COV2.S, Ad5-nCOV, and Gam-COVID-Vac were well tolerated throughout clinical development. ^{, , , , ,} Injection-site tenderness, ^{, ,} headache, ^, and fati-​gue ^{, ,} were among the most frequently reported local and systematic reactions in Phase III studies. The overall reactogenicity profile has been shown to be more tolerable with lower doses of vaccine, ^, in individuals with increasing age, ^, and following a second dose. ^{, ,}

Efficacy of Adenovirus-Based COVID-19 Vaccines

The clinical profile observed in multiple Phase III studies has supported the licensure of several adenovirus-based COVID-19 vaccines that have demonstrated efficacy in preventing symptomatic SARS-CoV-2 illness (see Table 68.2 ), enabling the vaccination of billions of individuals globally within the first year of deployment. ^, Vaccine efficacy from adenovirus-based COVID-19 vaccines has been observed across diverse trial populations ^{, , ,} including older adults, and those with comorbidities such as diabetes, cardiovascular, and respiratory disease. ^{, ,} The efficacy of AZD1222 has been demonstrated in multiple randomized controlled clinical trials. ^{, ,} An initial interim analysis pooling study in the United Kingdom, Brazil, and South Africa demonstrated an initial efficacy of 70.4% in preventing symptomatic SARS-CoV-2 infection and supported initial emergency use authorization in the United Kingdom. A primary analysis from a Phase III study in the United States, Peru, and Chile estimated an overall efficacy against symptomatic disease of 74.0%. Efficacy of AZD1222 at preventing infection with SARS-CoV-2 (as measured by nucleocapsid antibody seroconversion of all participants who tested positive for SARS-CoV-2 regardless of symptoms or severity) was estimated at 64.3%. Single-dose Ad26.COV2.S showed overall efficacy in preventing moderate-to-severe/critical infection of 66.9% at 14 days postimmunization and 66.1% at 28 days postimmunization. An interim analysis of a Phase III study of Gam-COVID-Vac—a heterologous 21-day prime-boost of Ad26 (prime) and Ad5 (boost)—demonstrated an efficacy of 91.6% against symptomatic infection 21 days postdose 1 (Ad26), that is, on the day of dose 2 (Ad5), AZD1222, ^, Ad26.COV.S, and Gam-COVID-Vac were highly protective against severe disease, hospitalization, and death during their respective Phase III studies.

The rapid vaccination rollout in response to the pandemic has provided invaluable insights on the effectiveness of adenovirus-based COVID-19 vaccines. For example, multiple studies have estimated the effectiveness of two doses of AZD1222 at preventing symptomatic infection as 77–89%. Emerging data also suggest that vaccination may reduce disease transmission by 30–47%. ^, The emergence of new variants of SARS-CoV-2 possessing mutations in the S glycoprotein remains a focus of significant international attention following early observations of increased transmissibility with SARS-CoV-2 B.1.1.7/Alpha ^182,186 and Delta, ^, reduced vaccine efficacy against mild-to-moderate disease with SARS-CoV-2 B.1.351/Beta, ^, and potential immune evasion mutations in B.1.1.28/Gamma variant/P.1 VoCs ^, (see Table 68.2 ). While investigations of vaccine effectiveness against VoC are ongoing, insights from real-world effectiveness studies suggest that existing COVID-19 vaccines may still confer adequate protection against severe disease, hospitalization, and death. The emergence of SARS-CoV-2 VoC emphasizes that ongoing vaccine development and vaccine re-engineering may be required for the duration of the pandemic.