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/Objectives: Fecal occult blood testing (FOBT) is a cornerstone of colorectal cancer (CRC) screening. The current quantitative FOBTs, however, were centralized, which limited their accessibility in preliminary screening implemented by community health. This study aimed to establish a rapid, highly sensitive quantitative FOB-detection method based on near-infrared fluorescence immunochromatography, hereinafter referred to as point-of-care fecal immunochemical testing (POCT-FIT), and to evaluate its performance against a centralized testing system in population-based CRC screening. Methods: Anti-human hemoglobin monoclonal antibodies were labeled with near-infrared fluorescent nanospheres (excitation 770 nm; emission 794 nm), and FOB test strips were prepared using immunochromatography. A fluorescence immunochromatography analyzer was used to scan the fluorescence intensities of the test line (T) and control line (C). The ratio of the fluorescence intensity on the T-line to that on the C one (T/C) was calculated at different hemoglobin concentrations, and a standard curve was generated over the span of 50 to 2000 ng/mL. A total of 115 clinical fecal samples were collected for methodological comparison with the commercially available OC-Sensor system (Eiken Chemical Co., Ltd., Tokyo, Japan). In a non-paired CRC screening program implemented by community health in Wenzhou, 3163 participants were assigned to the centralized testing group (OC-Sensor system) and 5211 participants to the POCT-FIT group (near-infrared fluorescence immunochromatography method). A positive threshold was set up at 100 ng/mL. Colonoscopy and histopathological results served as the gold standard. Results: The testing results of the two groups for the 115 samples showed that there is a strong linear correlation (R = 0.9959) and good agreement between the two methods. In the screening of community health, CRC prevalence was 2.78% (88/3163) in the centralized group (OC-Sensor) and 1.00% (52/5211) in the decentralized one (POCT-FIT). Sensitivities for CRC were 98.9% (95% CI: 95.8–99.9%) and 100% (95% CI: 93.2–100%), respectively, with no statistically significant difference; specificities were 29.2% (95% CI: 27.6–30.8%) and 27.3% (95% CI: 26.1–28.5%), with the centralized group being slightly higher (p = 0.001). Positive and negative likelihood ratios were comparable between the two methods. Conclusions: The sensitivity of the POCT-FIT method was comparable to that of the OC-Sensor method, and its negative results were associated with a lower probability of missed CRC, which may help reduce medical insurance expenditure for CRC treatment. POCT-FIT is simple to operate and portable, and the price of the disposable immunochromatographic test strip is much lower than that of the turbidimetric method (centralized testing system). Thus, it meets the practical needs of settings with low daily testing numbers (such as preliminary community health or resource-limited and sparsely populated areas) and serves as an effective complement to centralized testing.

Background/Objectives: In previous studies, we demonstrated that the HIV-1 Env-Gag VLP mRNA vaccine elicited superior cellular immune responses. In this study, we further evaluated the immunogenicity of the Env-Gag VLP mRNA and adenovirus vector vaccines when administered individually or in combination in mice. Methods: BALB/c mice were divided into four groups and immunized twice at a 3-week interval. The three groups received either the Env-Gag VLP mRNA vaccine, the adenovirus vector vaccines expressing env and gag genes, or PBS as a control. The fourth group received a prime-boost regimen, primed with the Env-Gag mRNA vaccine and boosted with the adenovirus vector vaccines. The HIV-1 specific cellular and humoral immune responses were measured 1, 2, 4 and 8 weeks after the last immunization. Results/Conclusions: The results showed that the mRNA vaccines prime-adenovirus vector vaccines boost elicited higher cellular immune responses than those induced by homologous regimens at multiple time points, especially 8 weeks after the last immunization. Although the level of gp120 binding antibody in the combined immunization group is significantly lower than that of in the VLP mRNA vaccine group, a more balanced Th1/Th2 responses were induced in the combined immunization group, and significantly higher and longer-lasting neutralizing antibody levels were detected in this group making it a very promising HIV vaccine strategy.