Boosting with variant-matched adenovirus-based vaccines promotes neutralizing antibody responses against SARS-CoV-2 Omicron sublineages in mice.

Global spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Omicron subvariants, such as BA.4, BA.5 and XBB.1.5, has been leading the recent wave of coronavirus disease 2019 (COVID-19). Unique mutations in the spike proteins of these emerging Omicron subvariants caused immune evasion from the pre-existing protective immunity induced by vaccination or natural infection. Previously, we developed AdCLD-CoV19-1, a non-replicating recombinant adenoviral vector that encodes the receptor binding domain of the spike protein of the ancestral SARS-CoV-2 strain. Based on the same recombinant adenoviral vector platform, updated vaccines that cover unique mutations found in each Omicron subvariant, including BA.1, BA.2, BA.4.1 and BA.5, were constructed. Preclinical studies revealed that each updated vaccine as a booster shot following primary vaccination targeting the ancestral strain improved neutralizing antibody responses against the pseudovirus of its respective strain most effectively. Of note, boosting with a vaccine targeting the BA.1 or BA.2 Omicron subvariant was most effective in neutralization against the pseudovirus of the BA.2.75 strain, whereas BA.4.1/5-adapted booster shots were most effective in neutralization against the BQ.1, BQ1.1 and BF.7 strains. Therefore, it is imperative to develop a vaccination strategy that can cover the unique spike mutations of currently circulating Omicron subvariants in order to prevent the next wave of COVID-19.

SARS-CoV-2 S1 Subunit Booster Vaccination Elicits Robust Humoral Immune Responses in Aged Mice.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has raised concerns about reduced vaccine effectiveness and the increased risk of infection, and while repeated homologous booster shots are recommended for elderly and immunocompromised individuals, they cannot completely protect against breakthrough infections. In our previous study, we assessed the immunogenicity of an adenovirus-based vaccine expressing SARS-CoV-2 S1 (Ad5.S1) in mice, which induced robust humoral and cellular immune responses (E. Kim, F. J. Weisel, S. C. Balmert, M. S. Khan, et al., Eur J Immunol 51:1774-1784, 2021, https://doi.org/10.1002/eji.202149167). In this follow-up study, we found that the mice had high titers of anti-S1 antibodies 1 year after vaccination, and one booster dose of the nonadjuvanted rS1Beta (recombinant S1 protein of SARS-CoV-2 Beta [B.1.351]) subunit vaccine was effective at stimulating strong long-lived S1-specific immune responses and inducing significantly high neutralizing antibodies against Wuhan, Beta, and Delta strains, with 3.6- to 19.5-fold increases. Importantly, the booster dose also elicited cross-reactive antibodies, resulting in angiotensin-converting enzyme 2 (ACE2) binding inhibition against spikes of SARS-CoV-2, including Omicron variants, persisting for >28 weeks after booster vaccination. Interestingly, the levels of neutralizing antibodies were correlated not only with the level of S1 binding IgG but also with ACE2 inhibition. Our findings suggest that the rS1Beta subunit vaccine candidate as a booster has the potential to offer cross-neutralization against broad variants and has important implications for the vaccine control of newly emerging breakthrough SARS-CoV-2 variants in elderly individuals primed with adenovirus-based vaccines like AZD1222 and Ad26.COV2.S. IMPORTANCE Vaccines have significantly reduced the incidences of severe coronavirus disease 2019 (COVID-19) cases and deaths. However, the emergence of SARS-CoV-2 variants has raised concerns about their increased transmissibility and ability to evade neutralizing antibodies, especially among elderly individuals who are at higher risks of mortality and reductions of vaccine effectiveness. To address this, a heterologous booster vaccination strategy has been considered as a solution to protect the elderly population against breakthrough infections caused by emerging variants. This study evaluated the booster effect of an S1 subunit vaccine in aged mice that had been previously primed with adenoviral vaccines, providing valuable preclinical evidence for elderly people vaccinated with the currently approved COVID-19 vaccines. This study confirms the potential for using the S1 subunit vaccine as a booster to enhance cross-neutralizing antibodies against emerging variants of concern.

Measurement of Adenovirus-Based Vector Heterogeneity.

Adenovirus vectors have become an important class of vaccines with the recent approval of Ebola and COVID-19 products. In-process quality attribute data collected during Adenovirus vector manufacturing has focused on particle concentration and infectivity ratios (based on viral genome: cell-based infectivity), and data suggest only a fraction of viral particles present in the final vaccine product are efficacious. To better understand this product heterogeneity, lab-scale preparations of two Adenovirus viral vectors, (Chimpanzee adenovirus (ChAdOx1) and Human adenovirus Type 5 (Ad5), were studied using transmission electron microscopy (TEM). Different adenovirus morphologies were characterized, and the proportion of empty and full viral particles were quantified. These proportions showed a qualitative correlation with the sample's infectivity values. Liquid chromatography-mass spectrometry (LC-MS) peptide mapping was used to identify key adenovirus proteins involved in viral maturation. Using peptide abundance analysis, a ∼5-fold change in L1 52/55k abundance was observed between low-(empty) and high-density (full) fractions taken from CsCl ultracentrifugation preparations of ChAdOx1 virus. The L1 52/55k viral protein is associated with DNA packaging and is cleaved during viral maturation, so it may be a marker for infective particles. TEM and LC-MS peptide mapping are promising higher-resolution analytical characterization tools to help differentiate between relative proportions of empty, non-infectious, and infectious viral particles as part of Adenovirus vector in-process monitoring, and these results are an encouraging initial step to better differentiate between the different product-related impurities.

Analyzing the immunogenicity of bivalent booster vaccinations in healthcare workers: The SWITCH ON trial protocol.

Vaccination against coronavirus disease 2019 (COVID-19) has contributed greatly to providing protection against severe disease, thereby reducing hospital admissions and deaths. Several studies have reported reduction in vaccine effectiveness over time against the Omicron sub-lineages. However, the willingness to receive regular booster doses in the general population is declining. To determine the need for repeated booster vaccinations in healthy individuals and to aid policymakers in future public health interventions for COVID-19, we aim to gain insight into the immunogenicity of the additional bivalent booster vaccination in a representative sample of the healthy Dutch population. The SWITCH ON study was initiated to investigate three main topics: i) immunogenicity of bivalent vaccines after priming with adenovirus- or mRNA-based vaccines, ii) immunological recall responses and reactivity with relevant variants after booster vaccination, and iii) the necessity of booster vaccinations for the healthy population in the future. Clinical trial registration: https://clinicaltrials.gov/, identifier NCT05471440.

SARS-Cov-2 incubation period according to vaccination status during the fifth COVID-19 wave in a tertiary-care center in Spain: a cohort study.

BACKGROUND: The incubation period of an infectious disease is defined as the elapsed time between the exposure to the pathogen and the onset of symptoms. Although both the mRNA-based and the adenoviral vector-based vaccines have shown to be effective, there have been raising concerns regarding possible decreases in vaccine effectiveness for new variants and variations in the incubation period. METHODS: We conducted a unicentric observational study at the Hospital Universitari de Bellvitge, Barcelona, using a structured telephone survey performed by trained interviewers to estimate the incubation period of the SARS-CoV-2 Delta variant in a cohort of Spanish hospitalized patients. The distribution of the incubation period was estimated using the generalized odds-rate class of regression models. RESULTS: From 406 surveyed patients, 242 provided adequate information to be included in the analysis. The median incubation period was 2.8 days (95%CI: 2.5-3.1) and no differences between vaccinated and unvaccinated patients were found. Sex and age are neither shown not to be significantly related to the COVID-19 incubation time. CONCLUSIONS: Knowing the incubation period is crucial for controlling the spread of an infectious disease: decisions on the duration of the quarantine or on the periods of active monitoring of people who have been at high risk of exposure depend on the length of the incubation period. Furthermore, its probability distribution is a key element for predicting the prevalence and the incidence of the disease.

Analysis of Host Immunological Response of Adenovirus-Based COVID-19 Vaccines.

During the SARS-CoV-2 global pandemic, several vaccines, including mRNA and adenovirus vector approaches, have received emergency or full approval. However, supply chain logistics have hampered global vaccine delivery, which is impacting mass vaccination strategies. Recent studies have identified different strategies for vaccine dose administration so that supply constraints issues are diminished. These include increasing the time between consecutive doses in a two-dose vaccine regimen and reducing the dosage of the second dose. We consider both of these strategies in a mathematical modeling study of a non-replicating viral vector adenovirus vaccine in this work. We investigate the impact of different prime-boost strategies by quantifying their effects on immunological outcomes based on simple system of ordinary differential equations. The boost dose is administered either at a standard dose (SD) of 1000 or at a low dose (LD) of 500 or 250 vaccine particles. Results show dose-dependent immune response activity. Our model predictions show that by stretching the prime-boost interval to 18 or 20, in an SD/SD or SD/LD regimen, the minimum promoted antibody (Nab) response will be comparable with the neutralizing antibody level measured in COVID-19 recovered patients. Results also show that the minimum stimulated antibody in SD/SD regimen is identical with the high level observed in clinical trial data. We conclude that an SD/LD regimen may provide protective capacity, which will allow for conservation of vaccine doses.

Performance of Homologous and Heterologous Prime-Boost Immunization Regimens of Recombinant Adenovirus and Modified Vaccinia Virus Ankara Expressing an Ag85B-TB10.4 Fusion Protein against Mycobacterium tuberculosis.

Tuberculosis (TB) remains a serious health issue around the word. Adenovirus (Ad)-based vaccine and modified vaccinia virus Ankara (MVA)-based vaccine have emerged as two of the most promising immunization candidates over the past few years. However, the performance of the homologous and heterologous prime-boost immunization regimens of these two viral vector-based vaccines remains unclear. In the present study, we constructed recombinant Ad and MVA expressing an Ag85B-TB10.4 fusion protein (AdH4 and MVAH4) and evaluated the impact of their different immunization regimens on the humoral and cellular immune responses. We found that the viral vector-based vaccines could generate significantly higher levels of antigen-specific antibodies, IFN-γ-producing splenocytes, CD69⁺CD8⁺ T cells, and IFN-γ secretion when compared with bacillus Calmette-Guérin (BCG) in a mouse model. AdH4-containing immunization regimens (AdH4-AdH4, AdH4-MVAH4, and MVAH4-AdH4) induced significantly stronger antibody responses, much more IFN-γ-producing splenocytes and CD69⁺CD8⁺ T cells, and higher levels of IFN-γ secretion when compared with the MVAH4-MVAH4 immunization regimen. The number of IFN-γ-producing splenocytes sensitive to CD8⁺ T-cell restricted peptides of Ag85B (9-1p and 9-2p) and Th1-related cytokines (IFN-γ and TNF-α) in the AdH4-MVAH4 heterologous prime-boost regimen immunization group was significantly higher than that in the other viral vector-based vaccine- and BCG-immunized groups, respectively. These results indicate that an immunization regimen involving AdH4 may have a higher capacity to induce humoral and cellular immune responses against TB in mice than that by regimens containing BCG or MVAH4 alone, and the AdH4-MVAH4 prime-boost regimen may generate an ideal protective effect.

Respiratory macrophages regulate CD4 T memory responses to mucosal immunization with recombinant adenovirus-based vaccines.

Respiratory immunization is an attractive way to generate systemic and mucosal protective memory responses that are required for preventing mucosally transmitted infections. However, the molecular and cellular mechanisms for controlling memory T cell responses remain incompletely understood. In this study, we investigated the role of respiratory macrophage (MΦ) in regulating CD4 T cell responses to recombinant adenovirus-based (rAd) vaccines. We demonstrated that rAd intranasal (i.n.) vaccination induced migration and accumulation of respiratory MΦ and circulatory monocytes in the mediastinal lymph nodes and lung parenchyma. Under the influence of respiratory MΦ CD4 T cells exhibited slow proliferation kinetics and an increased tendency of generating central memory, as opposed to effector memory, CD4 T cell responses in vitro and in vivo. Correspondingly, depletion of MΦ using clodronate-containing liposome prior to i.n. immunization significantly enhanced CD4 T cell proliferation and increased the frequency of CD4 memory T cells in the airway lumen, demonstrating that MΦ initially serve as a negative regulator in limiting generation of mucosal tissue-resident memory CD4 T cells. However, clodronate-containing liposome delivery following i.n. immunization markedly reduced the frequencies of memory CD4 T cells in the airway lumen and spleen, indicating that respiratory MΦ and potentially circulating monocytes are critically required for maintaining long-term memory CD4 T cells. Collectively, our data demonstrate that rAd-induced mucosal CD4 T memory responses are regulated by respiratory MΦ and/or monocytes at multiple stages.