To investigate the impact of different 5ʹ untranslated regions (UTRs) on mRNA vaccine translation efficiency, five dual-reporter gene expression plasmids with different 5ʹUTRs were constructed. The corresponding mRNA transcripts were transcribed and capped in vitro. By comparing the expression levels of reporter genes with different 5ʹUTRs, we identified the 5ʹUTR associated with the highest expression level. Subsequently, HIVgp145 mRNA vaccines containing various 5ʹUTRs were constructed and verified. The results demonstrated that mRNA 3 (β-globin 5ʹUTR) displayed the greatest number of green fluorescence-positive cells and the highest luciferase fluorescence intensity in the reporter gene expression system. Further, among the HIVgp145 mRNA vaccines with different 5ʹUTRs, mRNA 7 (β-globin 5ʹUTR) exhibited the highest level of expression. These findings indicate that it is feasible to use the 5ʹUTR of β-globin in an mRNA vaccine, laying the foundation for animal immunogenicity testing.

The development of gene editing tools such as CRISPR-Cas9 and base editors (BE) is critical for genetic diseases and cancer. Lentivirus-like particles (LVLPs) grows into an auspicious platform for delivering mRNA or ribonucleic proteins (RNPs) due to it integrates the advantage of viral and non-viral vectors. Current LVLP systems predominantly utilize HIV-Gag and Pol proteins. However, the reverse transcriptase and integrase of Pol, pose risks of genomic integration and potential tumorigenesis. Enhancing the safety of VLP system is essential. This study focuses on improving the LVLP to minimize these risks. We implemented a Gag-Only strategy, constructing LVLPs with HIV-Gag protein, thereby eliminating the integration risks linked to Pol. By leveraging the interactions between MS2-MCP (MS2 coat protein), PP7 and PP7 BP (PP7 binding protein), and the psi (HIV packaging signal) with HIV-Gag, we encapsulated PAMless andesine base editor (CE-8e-SpRY) mRNA and sgRNA targeting the PD1 start codon (ATG) into the LVLP. Using recombinant lentiviral vector technology, we developed a stable PD1-expressing 293T cell line (PD1-293T) to assess the editing efficiency of LVLP. The psi-LVLP demonstrated effective packaging capabilities, achieving 15% base editing efficiency in 293T cells. By optimizing plasmid ratios, we observed increased CE-8e-SpRY mRNA copy numbers, with 30% base editing efficiency. Additionally, the integration of HDVrz (hepatitis delta virus ribozyme) and psi into sgRNA (HDVrz-psi-LVLP) substantially enhanced sgRNA copy numbers, resulting in approximately 50% base editing efficiency in 293T cells and 20% base editing efficiency in Jurkat cells. Mendelian randomization analyses revealed significant genetic correlations between PD1, B2M, CIITA, and TIGIT genes with various cancer risks. Furthermore, HDVrz-psi-LVLPs targeting the start codons of B2M, CIITA, and TIGIT exhibited high base editing activity in both Jurkat and 293T cells. In conclusion, this optimized platform effectively encapsulates CE-8e-SpRY mRNA and sgRNA, achieving high editing efficiencies across multiple genes and cell types. We introduce a safer and more efficient gene editing tool delivery system by constructing LVLPs based on the Gag-Only strategy. Our study presents a promising implication for cancer immunotherapy.

Background/Objectives: Developing durable cellular immunity remains a critical challenge for HIV vaccine development. Methods: We evaluated a sequential and repeated heterologous prime–boost vaccination regimen using four distinct vector-based vaccines (DNA, rAd5, rSeV, and rMVA) expressing HIV-1 gag in rhesus macaques over a decade-long observation period. Results: Compared to the two-vector and control groups, the four-vector regimen elicited potent gag-specific cellular immune responses, as evidenced by IFN-γ ELISPOT assays showing sustained responses exceeding 500 SFCs/10⁶ PBMCs for up to 52 or 69 weeks post-vaccination. Intracellular cytokine staining revealed multifunctional CD4+ and CD8+ T-cell responses, while humoral immunity against Ad5 vectors remained manageable despite repeated administrations. Conclusions: These findings demonstrate that sequential and repeated heterologous vaccination effectively induces and maintains durable cellular immunity, providing a strategic framework for HIV vaccine design.

Background: The development of a protective vaccine is critical for conclusively ending the human immunodeficiency virus (HIV) epidemic. Methods: We constructed nucleotide-modified mRNA vaccines expressing HIV-1 Env and Gag proteins. Env–gag virus-like particles (VLPs) were generated through co-transfection with env and gag mRNA vaccines. BALB/c mice were immunized with env mRNA, env–gag VLP mRNA, env plasmid DNA vaccine, or lipid nanoparticle (LNP) controls. HIV Env-specific binding and neutralizing antibodies in mouse sera were assessed via enzyme-linked immunosorbent assay (ELISA) and pseudovirus-based neutralization assays, respectively. Env-specific cellular immune responses in mouse splenocytes were evaluated using an Enzyme-linked immunosorbent assay (ELISpot) and in vivo cytotoxic T cell-killing assays. Results: The Env-specific humoral and cellular immune responses elicited by HIV-1 env mRNA and env–gag VLP mRNA vaccine were stronger than those induced by the DNA vaccine. Specific immune responses induced by the env mRNA vaccine were significantly stronger in the high-dose group than in the low-dose group. Immunization with co-formulated env and gag mRNAs elicited superior cellular immune responses compared to env mRNA alone. Conclusions: These findings suggest that the env–gag VLP mRNA platform holds significant promise for HIV-1 vaccine development.

Considering the absence of a widely utilized EBV vaccine, we have developed an EBV gL-gH344-Ferritin nanoparticle vaccine utilizing ferritin as a carrier. The gL-gH344-Ferritin fusion gene was synthesized and inserted into the pET30a plasmid. The expression of the fusion protein in the recombinant plasmid was verified by Western blot. Then, the gL-gH344-Ferritin subunit nanoparticle vaccine was obtained by purification of the fusion protein. BALB/c mice were immunized using a two-dose protocol. The titers of EBV specific antibodies were determined using enzyme-linked immunosorbent assay at 4, 8, and 12 weeks after the initial immunization. Moreover, the levels of EBV gL-gH344 specific splenocytes secreting interferon (IFN)-γ and interleukin (IL)-6 were determined using an enzyme-linked immunospot assay. The pET30a-gL-gH344-Ferritin prokaryotic expression plasmid was successfully constructed. gL-gH344-Ferritin was efficiently expressed in E. coli. Following immunization with gL-gH344-Ferritin, the mice sera demonstrated elevated titers of EBV specific immunoglobulin G. Moreover, after stimulating with EBV gL-gH344 specific peptides, the splenocytes of the immunized mice showed a marked tendency to secrete large amounts of IFN-γ and IL-6. The gL-gH344-Ferritin nanoparticle vaccine carrying the EBV gL-gH344 fusion protein induced robust and sustained specific humoral and cellular immune responses in mice.

Background: Developing an effective vaccine is crucial for the prevention and control of AIDS. Viral vector-based vaccines, particularly those utilizing homologous or heterologous prime-boost strategies, represent an important direction in current HIV vaccine research. Methods: In this study, replication-defective chimeric adenovirus Ad5F35 and modified vaccinia virus Ankara (rMVA) vector vaccines expressing the HIV-1 AEgp145 were successfully constructed, designated as Ad5F35-AEgp145 and rMVA-AEgp145, respectively. Sixty BALB/c mice were randomly divided into three groups: Ad5F35 alone, rMVA prime/Ad5F35 boost, and PBS control. The mice were immunized intramuscularly at weeks 0 and 3, and humoral and cellular immune responses were assessed at 4, 8, 12, and 16 weeks after the initial immunization. Results: The homologous Ad5F35 and heterologous rMVA/Ad5F35 vaccination regimens elicited comparable levels of HIV Env-specific cellular immune responses, peaking at 2100 ± 222 SFCs/million splenocytes and 2200 ± 619 SFCs/million splenocytes, respectively (p > 0.05). Compared to the heterologous regimen, the homologous Ad5F35 regimen induced significantly higher levels of gp120-binding antibodies at weeks 4 and 8 post-initial immunization, with geometric mean titers of 1:25,600 ± 7011 versus 1:1280 ± 150.7 and 1:10,240 ± 4048 versus 1:2560 ± 391.9, respectively. Furthermore, neutralizing activity at week 8 was significantly higher in the homologous group, with a 50% neutralization titers of 1:45 compared to 1:12 in the heterologous group (p < 0.01). Conclusion: This study demonstrates that the Ad5F35-AEgp145 vaccine, whether administered alone or in combination with rMVA-AEgp145, effectively induces strong and comparable cellular immune responses targeting HIV-1 Env in mice. While both regimens are effective, homologous immunization elicits moderately higher levels of antibody responses. These findings provide an important foundation for the further investigation of vector-based HIV vaccine formulations.