T-cell immunity plays an important role in the control of SARS-CoV-2 and has a great cross-protective effect on the variants. The Omicron BA.1 variant contains more than 30 mutations in the spike and severely evades humoral immunity. To understand how Omicron BA.1 spike mutations affect cellular immunity, the T-cell epitopes of SARS-CoV-2 wild-type and Omicron BA.1 spike in BALB/c (H-2^d) and C57BL/6 mice (H-2^b) were mapped through IFNγ ELISpot and intracellular cytokine staining assays. The epitopes were identified and verified in splenocytes from mice vaccinated with the adenovirus type 5 vector encoding the homologous spike, and the positive peptides involved in spike mutations were tested against wide-type and Omicron BA.1 vaccines. A total of eleven T-cell epitopes of wild-type and Omicron BA.1 spike were identified in BALB/c mice, and nine were identified in C57BL/6 mice, only two of which were CD4⁺ T-cell epitopes and most of which were CD8⁺ T-cell epitopes. The A67V and Del 69-70 mutations in Omicron BA.1 spike abolished one epitope in wild-type spike, and the T478K, E484A, Q493R, G496S and H655Y mutations resulted in three new epitopes in Omicron BA.1 spike, while the Y505H mutation did not affect the epitope. These data describe the difference of T-cell epitopes in SARS-CoV-2 wild-type and Omicron BA.1 spike in H-2^b and H-2^d mice, providing a better understanding of the effects of Omicron BA.1 spike mutations on cellular immunity.

The modified vaccinia virus Ankara (MVA) is approved for use as a smallpox and monkeypox virus vaccine and was also designed as a popular recombinant viral vector for vaccine development and gene therapy. However, the extensive genomes of poxviruses present a significant challenge for the development of recombinant viral vaccines; therefore, it is essential to establish a user-friendly in vitro reverse genetic system. We systematically assembled the 180-kb MVA genome into a five-plasmid system, facilitating one-step packaging of the MVA virus. The MVA rescued using this system exhibited similar virological characteristics, including host cell tropism, growth kinetics, plaque size, and viral particles, comparable to those of wild-type MVA. Immunization with rescued MVA intramuscularly or subcutaneously triggered robust-specific immune responses and conferred protection against lethal attacks by the ectromelia virus in mice. We also developed a recombinant MVA-Luc-eGFP virus, which served as a tool for screening antiviral compounds against poxviruses. The synthetic MVA system efficiently generates recombinant vaccines with robust immune responses. These findings provide a novel and fast method for engineering large viral genomes with more specialized structures and lay a foundation for the advancement of more rapid and effective viral vector vaccines.

Introduction: Adenovirus (Ad) vectors are widely used for gene delivery, and some of them have been approved for vaccine development. In particular, the recombinant COVID-19 vaccine for inhalation, which was developed using adenovirus type 5 (Ad5), represents a milestone in respiratory immunization. Owing to the high pre-existing immunity (PEI) to Ad5, the development of an adenoviral vector with lower PEI and higher immunogenicity has been explored. However, the majority of the developed novel Ad vectors showed suboptimal immunogenicity compared to Ad5 in animal models.Method: In this study, we constructed a novel replication-deficient viral vector based on human adenovirus type 4 (Ad4), which has long been used as a live virus vaccine with a favorable safety profile in the U.S. military. The mice were immunized intramuscularly or intranasally with an Ad4-vectored vaccine to verify immune responses and protective efficacy.Results: Compared with Ad5, the novel Ad4 vector showed comparable viral growth kinetics and transgene expression in cells and similar exogenous protein expression and distribution in mice. Furthermore, the Ad4-vectored vaccine elicited superior humoral and cellular responses and protective effects when vaccinated intranasally than those triggered by the Ad5-vectored vaccine. Finally, the heterologous Ad5 prime and Ad4 boost immunization showed better immunogenicity and protective efficacy.Discussion: This study broadens the research trajectory of adenovirus-vectored vaccines and offers a new option for the development of recombinant viral-vectored vaccines.

Mycoplasma pneumoniae (MP), a major cause of community-acquired pneumonia, poses an escalating public health concern due to the increasing prevalence of ma