Replication incompetent human adenovirus serotype 5 (HAdV-C5) has been extensively used as a delivery vehicle for gene therapy proteins and infectious disease antigens. These vectors infect replicating and nonreplicating cells, have a broad tissue tropism, elicit high immune responses and are easily purified to high titers. However, the utility of HAdV-C5 vectors as potential vaccines is limited due to pre-existing immunity within the human population that significantly reduces the immunogenicity of HAdV-C5 vaccines. In recent years, adenovirus vaccine development has focused on simian-derived adenoviral vectors, which have the desirable vector characteristics of HAdV-C5 but with negligible seroprevalence in the human population. Here, we discuss recent advances in simian adenovirus vaccine vector development and evaluate current research specifically focusing on clinical trial data.

Seasonal booster vaccination is the primary intervention for protection from respiratory viral infections, such as influenza virus or SARS-CoV-2. However, efficacy is often limited because immune exposure to prior strains impairs development of new responses. In this study, we sought to determine how this issue could be overcome in a mouse model of heterologous immunization against WT and omicron strains of SARS-CoV-2. Intranasal booster immunization circumvented the shortcomings of intramuscular immunization, resulting in superior systemic and mucosal T and B cell immunity and better viral control following SARS-CoV-2 challenge in hamsters. Mechanistically, an intranasal omicron booster immunization bypassed deleterious immune imprinting following intramuscular ancestral strain prime, which allowed for induction of de novo lung B cell and antibody responses against the omicron strain. Cross-reactive memory T cells were also efficiently recruited into the lungs. These findings support further testing of mucosal booster vaccines against respiratory viruses, particularly as a means of simultaneously overcoming deleterious immunological imprinting and enhancing mucosal responses.

Background: Adenovirus serotype 5 (Ad5) has many favourable characteristics for development as a gene therapy vector. However, the utility of current Ad5 vectors is limited by transient transgene expression, toxicity and immunogenicity. The most promising form of vector is the high capacity type, which is deleted for all viral genes. However, these vectors can only be produced to relatively low titres and with the aid of helper virus. Therefore a continuing challenge is the generation of more effective Ad5 vectors that can still be grown to high titres. Our approach is to generate complementing cell lines to support the growth of Ad5 vectors with novel late gene deficiencies. Results: We have used LoxP/Cre recombination mediated cassette exchange (RMCE) to generate cell lines expressing Ad5 proteins encoded by the L4 region of the genome, the products of which play a pivotal role in the expression of Ad5 structural proteins. A panel of LoxP parent 293 cell lines was generated, each containing a GFP expression cassette under the control of a tetracycline-regulated promoter inserted at a random genome location; the cassette also contained a LoxP site between the promoter and GFP sequence. Clones displayed a variety of patterns of regulation, stability and level of GFP expression. Clone A1 was identified as a suitable parent for creation of inducible cell lines because of the tight inducibility and stability of its GFP expression. Using LoxP-targeted, Cre recombinase-mediated insertion of an L4 cassette to displace GFP from the regulated promoter in this parent clone, cell line A1-L4 was generated. This cell line expressed L4 100K, 22K and 33K proteins at levels sufficient to complement L4-33K mutant and L4-deleted viruses. Conclusions: RMCE provides a method for rapid generation of Ad5 complementing cell lines from a pre-selected parental cell line, chosen for its desirable transgene expression characteristics. Parent cell lines can be selected for high or low gene expression, and for tight regulation, allowing viral protein expression to mirror that found during infection. Cell lines derived from a single parent will allow the growth of different vectors to be assessed without the complication of varying complementing protein expression.

Background/Objectives: The large size of commonly used regulatory elements such as the cytomegalovirus (CMV) immediate-early promoter imposes a significant burden on the already restricted payload capacity of first-generation adenoviral vectors, potentially hindering the development of multi-antigen vaccine candidates. To address this limitation, we have engineered a panel of novel, small, semi-synthetic promoters designed to leverage the changes in transcriptomic milieu following adenoviral vector entry. Methods: Eight synthetic enhancer modules (SE1–SE8) were designed in silico, each composed of transcription factor binding sites (TFBSs) previously found in host genes that are upregulated during early adenoviral infection. These synthetic enhancers were coupled with a minimal CMV core promoter to generate a panel of compact semi-synthetic promoters (cSE1–cSE8), and their activity was evaluated in the context of ChAdOx1 viral vectors expressing GFP or a modified Plasmodium falciparum circumsporozoite (CSN) antigen. Promoter performance was characterised in vitro via flow cytometry, RT-qPCR, and Western blotting, and in vivo by quantifying antigen-specific T-cell (IFN-γ ELISpot) and IgG antibody (ELISA) responses in BALB/c mice. Results: In vitro characterisation revealed a wide range of promoter activity across the panel, with cSE3 and cSE5 driving transgene expression levels comparable to the benchmark CMV promoters despite their markedly reduced genomic footprint. In vivo, ChAdOx1 vectors incorporating cSE3 and cSE5 elicited potent antigen-specific T-cell and IgG responses that were comparable to those induced by the larger CMV control promoters. Conclusions: We have successfully developed semi-synthetic promoters that match the potency of the much larger, frequently used CMV promoters whilst simultaneously reducing genomic footprint. These novel regulatory elements will facilitate the design of next-generation vaccines, particularly those requiring large antigens or multi-antigen cassettes.

During the COVID-19 pandemic, antibody-based vaccines targeting the SARS-CoV-2 spike glycoprotein were the key focus for development because neutralizing antibodies were associated with protection against the SARS-CoV-2 infection in pre-clinical models and human trials. While deploying these spike-based vaccines saved millions of lives worldwide, it has become clear that the immunological mechanisms of protection against severe disease are multifaceted and involve non-neutralizing antibody components. Here, we describe a novel pan-sarbecovirus T-cell vaccine, ChAdOx1.COVconsv12, designed to complement and broaden the protection of spike vaccines. The vaccine immunogen COVconsv12 employs the two regions in the viral proteome most conserved among sarbecoviruses delivered by replication-deficient vector ChAdOx1. It focuses T cells towards epitopes shared among sarbecoviruses including evolving SARS-CoV-2 variants. Here, we show that ChAdOx1.COVconsv12 induced broad T-cell responses in the BALB/c and C57BL/6 mice. In the Syrian hamster challenge model, ChAdOx1.COVconsv12 alone did not protect against the SARS-CoV-2 infection, but when co-administered with 1/50th of the ChAdOx1 nCoV-19 spike vaccine protective dose, faster recovery and lower oral swab viral load were observed. Induction of CD8+ T cells may decrease COVID-19 severity and extend the T-cell response coverage of variants to match the known (and as yet unknown) members of the beta-coronavirus family.