伊従 光洋

Evaluating long-term immunity after COVID-19 infection and vaccination is critical for managing potential outbreaks. The aim of this study was to develop a cost-effective in-house enzyme-linked immunosorbent assay (ELISA) based on Escherichia coli-expressed SARS-CoV-2 spike protein (E-S1) for antibody detection and to evaluate its performance. The system was validated by comparing the in-house ELISA results with those obtained using a commercial ELISA with HEK293-expressed spike protein (H-S1). Recombinant SARS-CoV-2 spike protein was produced in E. coli, purified, and validated for antigenicity via ELISA. Indirect ELISAs with both E-S1 and H-S1 antigens were performed on 386 serum samples from COVID-19 survivors, vaccinated individuals, and pre-pandemic controls collected at different time points. The E-S1 ELISA showed a statistically significant but weak correlation with H-S1 ELISA across all samples (r=0.205; p=0.0001). Stronger correlations were observed among vaccinated individuals with prior infection on day 90 (r=0.6017; p<0.001) and in naïve vaccine recipients on day 30 (r=0.5361; p=0.0003). Pre-pandemic sera from a rural population in Sumba Island exhibited high background reactivity in E-S1 ELISA, likely due to anti-E. coli antibodies, while urban pre-pandemic sera from Jakarta showed a stronger correlation with H-S1 ELISA. This suggests potential regional or immune background differences influencing assay performance. Although E-S1 retained antigenic properties, its diagnostic utility is limited by non-specific reactivity and reduced sensitivity compared to H-S1. In conclusion, E. coli expression systems may not be ideal for producing spike protein-based ELISA antigens specific to SARS-CoV-2. Alternative expression systems, such as human or baculovirus, could enhance diagnostic accuracy and specificity for COVID-19 antibody detection.

Background/Objectives: We developed a multistage Plasmodium falciparum vaccine using a heterologous prime-boost immunization strategy. This involved priming with a highly attenuated, replication-competent vaccinia virus strain LC16m8Δ (m8Δ) and boosting with adeno-associated virus type 1 (AAV1). This approach demonstrated 100% efficacy in both protection and transmission-blocking in a murine model. In this study, we compared our LC16m8∆/AAV1 vaccine, which harbors a gene encoding Pfs25-PfCSP fusion protein, to RTS,S/AS01 (RTS,S) in terms of immune responses, protective efficacy, and transmission-blocking activity (TBA) in murine models. Methods: Mice were immunized following prime-boost vaccine regimens m8∆/AAV1 or RTS,S and challenged with transgenic Plasmodium berghei parasites. Immune responses were assessed via ELISA, and TB efficacy was evaluated using direct feeding assays. Results: m8∆/AAV1 provided complete protection (100%) in BALB/c mice and moderate (40%) protection in C57BL/6 mice, similar to RTS,S. Unlike RTS,S’s narrow focus (repeat region), m8∆/AAV1 triggered antibodies for all PfCSP regions (N-terminus, repeat, and C-terminus) with balanced Th1/Th2 ratios. Regarding transmission blockade, serum from m8∆/AAV1-vaccinated BALB/c mice achieved substantial transmission-reducing activity (TRA = 83.02%) and TB activity (TBA = 38.98%)—attributes not observed with RTS,S. Furthermore, m8∆/AAV1 demonstrated durable TB efficacy (94.31% TRA and 63.79% TBA) 100 days post-immunization. Conclusions: These results highlight m8∆/AAV1′s dual action in preventing sporozoite invasion and onward transmission, a significant advantage over RTS,S. Consequently, m8∆/AAV1 represents an alternative and a promising vaccine candidate that can enhance malaria control and elimination strategies.

In the post-pandemic era, evaluating long-term immunity against COVID-19 has become increasingly critical, particularly in light of continuous SARS-CoV-2 mutations. This study aimed to assess the long-term humoral immune response in sera collected in Makassar. We measured anti-RBD IgG levels and neutralization capacity (NC) against both the Wild-Type (WT) Wuhan-Hu and Omicron XBB.1.5 variants across groups of COVID-19-vaccinated individuals with no booster (NB), single booster (SB), and double booster (DB). The mean durations since the last vaccination were 25.11 months, 19.24 months, and 16.9 months for the NB, SB, and DB group, respectively. Additionally, we evaluated the effect of breakthrough infection (BTI) history, with a mean duration since the last confirmed infection of 21.72 months. Our findings indicate fair long-term WT antibody (Ab) titers, with the DB group showing a significantly higher level than the other groups. Similarly, the DB group demonstrated the highest anti-Omicron XBB.1.5 Ab titer, yet it was insignificantly different from the other groups. Although the level of anti-WT Ab titers was moderate, we observed near-complete (96–97%) long-term neutralization against the WT pseudo-virus for all groups. There was a slight decrease in NC against Omicron XBB.1.5 compared to the WT among all groups, as DB group, SB group, and NB group showed 80.71 ± 3.9%, 74.29 ± 6.7%, and 67.2 ± 6.3% neutralization activity, respectively. A breakdown analysis based on infection and vaccine status showed that booster doses increase the NC against XBB.1.5, particularly in individuals without BTI. Individuals with BTI demonstrate a better NC compared to their counterpart uninfected individuals with the same number of booster doses. Our findings suggest that long-term immunity against SARS-CoV-2 persists and is effective against the mutant variant. Booster doses enhance the NC, especially among uninfected individuals.

Among Plasmodium spp. responsible for human malaria, Plasmodium vivax ranks as the second most prevalent and has the widest geographical range; however, vaccine development has lagged behind that of Plasmodium falciparum, the deadliest Plasmodium species. Recently, we developed a multistage vaccine for P. falciparum based on a heterologous prime-boost immunization regimen utilizing the attenuated vaccinia virus strain LC16m8Δ (m8Δ)-prime and adeno-associated virus type 1 (AAV1)-boost, and demonstrated 100% protection and more than 95% transmission-blocking (TB) activity in the mouse model. In this study, we report the feasibility and versatility of this vaccine platform as a P. vivax multistage vaccine, which can provide 100% sterile protection against sporozoite challenge and >95% TB efficacy in the mouse model. Our vaccine comprises m8Δ and AAV1 viral vectors, both harboring the gene encoding two P. vivax circumsporozoite (PvCSP) protein alleles (VK210; PvCSP-Sal and VK247; -PNG) and P25 (Pvs25) expressed as a Pvs25–PvCSP fusion protein. For protective efficacy, the heterologous m8Δ-prime/AAV1-boost immunization regimen showed 100% (short-term; Day 28) and 60% (long-term; Day 242) protection against PvCSP VK210 transgenic Plasmodium berghei sporozoites. For TB efficacy, mouse sera immunized with the vaccine formulation showed >75% TB activity and >95% transmission reduction activity by a direct membrane feeding assay using P. vivax isolates in blood from an infected patient from the Brazilian Amazon region. These findings provide proof-of-concept that the m8Δ/AAV1 vaccine platform is sufficiently versatile for P. vivax vaccine development. Future studies are needed to evaluate the safety, immunogenicity, vaccine efficacy, and synergistic effects on protection and transmission blockade in a non-human primate model for Phase I trials.