BACKGROUND: The recently circulating Omicron variants BA.2.86 and JN.1 were identified with more than 30 amino acid changes on the spike protein compared to BA.2 or XBB.1.5. This study aimed to comprehensively assess the immune escape potential of BA.2.86, JN.1, EG.5, and EG.5.1.
METHODS: We collected human and murine sera to evaluate serological neutralization activities. The participants received three doses of coronavirus disease 2019 (COVID-19) vaccines or a booster dose of the ZF2022-A vaccine (Delta-BA.5 receptor-binding domain [RBD]-heterodimer immunogen) or experienced a breakthrough infection (BTI). The ZF2202-A vaccine is under clinical trial study (ClinicalTrials.gov: NCT05850507). BALB/c mice were vaccinated with a panel of severe acute respiratory syndrome coronavirus 2 RBD-dimer proteins. The antibody evasion properties of these variants were analyzed with 41 representative human monoclonal antibodies targeting the eight RBD epitopes.
RESULTS: We found that BA.2.86 had less neutralization evasion than EG.5 and EG.5.1 in humans. The ZF2202-A booster induced significantly higher neutralizing titers than BTI. Furthermore, BA.2.86 and JN.1 exhibited stronger antibody evasion than EG.5 and EG.5.1 on RBD-4 and RBD-5 epitopes. Compared to BA.2.86, JN.1 further lost the ability to bind to several RBD-1 monoclonal antibodies and displayed further immune escape.
CONCLUSIONS: Our data showed that the currently dominating sub-variant, JN.1, showed increased immune evasion compared to BA.2.86 and EG.5.1, which is highly concerning. This study provides a timely risk assessment of the interested sub-variants and the basis for updating COVID-19 vaccines.
BACKGROUND: This work was funded by the National Key R&D Program of China, the National Natural Science Foundation of China, the Beijing Life Science Academy, the Bill & Melinda Gates Foundation, and the Postdoctoral Fellowship Program of China Postdoctoral Science Foundation (CPSF).

BACKGROUND: Safe and effective vaccines are crucial for the control and eventual elimination of malaria. Novel approaches to optimize and improve vaccine efficacy are urgently required. Nanoparticle-based delivery platforms are considered potent and powerful tools for vaccine development.
METHODS: In this study, we developed a transmission-blocking vaccine against malaria by conjugating the ookinete surface antigen PSOP25 to the Acinetobacter phage coat protein AP205, forming virus-like particles (VLPs) using the SpyTag/SpyCatcher adaptor system. The combination of AP205-2*SpyTag with PSOP25-SpyCatcher resulted in the formation of AP205-PSOP25 complexes (VLP-PSOP25). The antibody titers and avidity of serum from each immunization group were assessed by ELISA. Western blot and IFA were performed to confirm the specific reactivity of the elicit antisera to the native PSOP25 in Plasmodium berghei ookinetes. Both in vitro and in vivo assays were conducted to evaluate the transmission-blocking activity of VLP-PSOP25 vaccine.
RESULTS: Immunization of mice with VLP-PSOP25 could induced higher levels of high-affinity antibodies than the recombinant PSOP25 (rPSOP25) alone or mixtures of untagged AP205 and rPSOP25 but was comparable to rPSOP25 formulated with alum. Additionally, the VLP-PSOP25 vaccine enhanced Th1-type immune response with remarkably increased levels of IgG2a subclass. The antiserum generated by VLP-PSOP25 specifically recognizes the native PSOP25 antigen in P. berghei ookinetes. Importantly, antisera generated by inoculation with the VLP-PSOP25 could inhibit ookinete development in vitro and reduce the prevalence of infected mosquitoes or oocyst intensity in direct mosquito feeding assays.
CONCLUSIONS: Antisera elicited by immunization with the VLP-PSOP25 vaccine confer moderate transmission-reducing activity and transmission-blocking activity. Our results support the utilization of the AP205-SpyTag/SpyCatcher platform for next-generation TBVs development.

Brucellosis is a major threat to public health and animal husbandry. Several in vivo vertebrate models, such as mice, guinea pigs, and nonhuman primates, have been used to study Brucella pathogenesis, bacteria-host interactions, and vaccine efficacy. However, these models have limitations whereas the invertebrate Galleria mellonella model is a cost-effective and ethical alternative. The aim of the present study was to examine the invertebrate G. mellonella as an in vivo infection model for Brucella. Infection assays were employed to validate the fitness of the larval model for Brucella infection and virulence evaluation. The protective efficacy of immune sera was evaluated by pre-incubated with a lethal dose of bacteria before infection. The consistency between the mouse model and the larval model was confirmed by assessing the protective efficacy of two Brucella vaccine strains. The results show that G. mellonella could be infected by Brucella strains, in a dose- and temperature-dependent way. Moreover, this larval model can effectively evaluate the virulence of Brucella strains in a manner consistent with that of mammalian infection models. Importantly, this model can assess the protective efficacy of vaccine immune sera within a day. Further investigation implied that haemolymph played a crucial role in the protective efficacy of immune sera. In conclusion, G. mellonella could serve as a quick, efficient, and reliable model for evaluating the virulence of Brucella strains and efficacy of immune sera in an ethical manner.

Immunoassays are commonly used in disease diagnosis and vaccine evaluation but can be costly and time-consuming when confronted with multivalent targets, such as antisera containing antibodies to human papillomavirus (HPV), because of their limited ability to discriminate between multiple analytes in a single reaction well. This study describes the development of a high-throughput liquid chip system that combines immunoassay techniques and magnetic beads to allow the simultaneous screening and quantitative detection of antibodies to four types of HPV using the Luminex fluoroimmunoassay system. Groups of beads embedded with fluorescent dyes at various ratios were coated with optimized HPV capture antigens and demonstrated excellent dose-dependent response to four monoclonal antibodies used as reference standards. This assay is sensitive, accurate, repeatable, and simple to perform, enabling multiplex antibody detection with a high degree of orthogonality. The performance of the Luminex system was compared with conventional immunoassays for quantitative detection of quadrivalent HPV antibodies in antisera of mice immunized with five lots of HPV vaccines, verifying the accuracy and detection efficiency of the assay. This strategy is a promising approach to characterizing antibodies present in polyclonal antisera and has promising applications in research, clinical, and industrial settings, for example, streamlining vaccine efficacy trials and vaccine lot inspection and release procedures.

To investigate the cross-reactivity between the sera collected from Vaccinia Virus Tiantan Strain vaccinated rabbits and viral antigens of monkeypox virus.
Vaccinia viruses were prepared on chicken embryo fibroblasts (CEF) and Vero cells respectively named as CEF-VTT NVSI-1 and Vero-VTT NVSI-1. Rabbits were inoculated with a total of three doses of adjuvanted 1.3 × 108 PFU CEF-VTT NVSI-1 each dose or adjuvanted 3.9 × 107 PFU Vero-VTT NVSI-1 (Freunds complete adjuvant) via the subcutaneous route. We then performed the enzyme-linked immunosorbent assay (ELISA) and bio-layer interferometry (BLI) for determination of the binding activity and affinity of immune sera to five crucial surface antigens on monkeypox virus including A35, B6R, H3 and to corresponding homologous antigens A33R, B5 and L1R of vaccinia virus. For comparison, plaque reduction neutralizing tests were used to evaluate the neutralization of immune sera against vaccinia virus.
Both CEF-VTT NVSI-1 and Vero-VTT NVSI-1 vaccinations following planned schedule could induce neutralizing antibody titers greater than 1:2048 in rabbit sera. Binding antibodies targeting monkeypox viral antigens were confirmed by both indirect ELISA and BLI methods. Indirect ELISA for rabbit sera revealed 1:51200 binding antibody titers to A35/B6R/H3 monkeypox virus antigens while BLI tests yielded affinities at 2 × 10-6 to 8 × 10-7 between the sera and the three antigens. Similarly, such sera showed binding strength to vaccinia virus antigens A33R/B5/L1R consistent with that to three preceding monkeypox virus antigens. These results demonstrated the cross-reactivity between the sera of vaccinia virus vaccinated animals and monkeypox virus antigens. Traditional ELISA test and BLI method displayed a high consistency in antigen screening and they were further proved to correlate to the results of plaque reduction neutralizing test, which indicates that BLI could be utilized as an indirect alternative for assessment of neutralizing activity of samples in response to live virus.
Sera of vaccinia virus-vaccinated rabbits exhibited cross-reactivity with viral antigens of monkeypox virus. Potential in improving the accuracy of antigen discovery while reducing the lengthy work needed for the screening as BLI method possesses, it contributes greatly to the rapid preliminary evaluation of immune response generated by vaccines.

Senecavirus A (SVA) is a type of nonenveloped single-stranded, positive-sense RNA virus. The VP2 protein is a structural protein that plays an important role in inducing early and late immune responses of the host. However, its antigenic epitopes have not been fully elucidated. Therefore, defining the B epitopes of the VP2 protein is of great importance to revealing its antigenic characterization. In this study, we analyzed B-cell immunodominant epitopes (IDEs) of the VP2 protein from the SVA strain CH/FJ/2017 using the Pepscan approach and a bioinformatics-based computational prediction method. The following four novel IDEs of VP2 were identified: IDE1, 41TKSDPPSSSTDQPTTT56; IDE2, 145PDGKAKSLQELNEEQW160; IDE3, 161VEMSDDYRTGKNMPF175; and IDE4, 267PYFNGLRNRFTTGT280. Most of the IDEs were highly conserved among the different strains. To our knowledge, the VP2 protein is a major protective antigen of SVA that can induce neutralizing antibodies in animals. Here, we analyzed the immunogenicity and neutralization activity of four IDEs of VP2. Consequently, all four IDEs showed good immunogenicity that could elicit specific antibodies in guinea pigs. A neutralization test in vitro showed that the peptide-specific guinea pig antisera of IDE2 could neutralize SVA strain CH/FJ/2017, and IDE2 was identified as a novel potential neutralizing linear epitope. This is the first time VP2 IDEs have been identified by using the Pepscan method and a bioinformatics-based computational prediction method. These results will help elucidate the antigenic epitopes of VP2 and clarify the basis for immune responses against SVA. IMPORTANCE The clinical symptoms and lesions caused by SVA are indistinguishable from those of other vesicular diseases in pigs. SVA has been associated with recent outbreaks of vesicular disease and epidemic transient neonatal losses in several swine-producing countries. Due to the continuing spread of SVA and the lack of commercial vaccines, the development of improved control strategies is urgently needed. The VP2 protein is a crucial antigen on the capsids of SVA particles. Furthermore, the latest research showed that VP2 could be a promising candidate for the development of novel vaccines and diagnostic tools. Hence, a detailed exploration of epitopes in the VP2 protein is necessary. In this study, four novel B-cell IDEs were identified using two different antisera with two different methods. IDE2 was identified as a new neutralizing linear epitope. Our findings will help in the rational design of epitope vaccines and further understanding of the antigenic structure of VP2.

Epidemiologic investigations in recent years have shown that the detection rate of avian hepatitis E virus (HEV) in chicken flocks is increasing in China. Nevertheless, effective prevention and control measures are still lacking. In this study, specific pathogen-free (SPF) chicken serum against HEV was prepared using recombinant HEV open reading frames (ORF2 and ORF3) proteins as immunogens. An SPF chicken infection model was established by intravenous inoculation of chick embryos. Swab samples were collected at 7, 14, 21, and 28 d of age and used to detect avian HEV load, along with other indicators, by fluorescence quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) assay. The therapeutic effects on blocking vertical HEV transmission were observed, by using the methods of antibody application alone, mixed, or combined application of each of the 2 antibodies with type I interferon. The results showed that type I interferon alone or in combination with antiserum reduced the positive rate of HEV from 100 to 62.5% and 25%, respectively. However, the avian HEV-positivity rate was reduced to 75, 50, and 37.5% after type I interferon was used alone or in combination with antisera against ORF2 and ORF3, respectively. The inhibitory effect of type I interferon alone or in combination with an antiserum, on HEV replication was more significant in cells than in vivo. In this study, the inhibitory effect of type I interferon alone or in combination with an antiserum on avian HEV replication was observed in vitro and in vivo, providing the necessary technical reserve for disease prevention and control.

Klebsiella pneumoniae is a leading cause of severe infections in humans and animals, and the emergence of multidrug-resistant strains highlights the need to develop effective vaccines for preventing such infections. Live attenuated vaccines are attractive vaccine candidates available in the veterinary field. We recently characterized that the K. pneumoniae kbvR (Klebsiella biofilm and virulence regulator) mutant was a highly attenuated strain in the mice model. In the present study, the characterization, safety, and protective efficacy of ΔkbvR strain as a live attenuated vaccine were evaluated. The synthesis and activity of type 1 fimbriae were increased in the ΔkbvR strain. All mice inoculated by the subcutaneous route with 105, 106, and 107 colony-forming units (CFU) doses of the ΔkbvR strain survived. Subcutaneous immunization with two doses of 105 or 107 CFU ΔkbvR elicited a robust humoral immune response, and provided protection against the following K. pneumoniae intraperitoneal infection. The antisera of mice immunized with 105 CFU dose improved the opsonophagocytic ability and complement-mediated lysis not only to the same serotype strain but also to the different serotype strain. The passive transfer of antisera from 105 CFU dose-immunized mice provided protection against K. pneumoniae infection. Overall, our results suggest the great potential of the ΔkbvR strain as a novel vaccine candidate against K. pneumoniae infections in herds or humans.

Influenza remains one of the most prevalent viruses circulating amongst humans and has resulted in several pandemics. The prevention and control of H3N2 influenza is complicated by its propensity for evolution, which leads to vaccine mismatch and reduced vaccine efficacies. This study employed the strategy of serial passaging to compare the evolution of the human seasonal influenza strain A/Singapore/G2-31.1/2014(H3N2) in MDCK-SIAT1 versus primary chick embryo fibroblast (CEF) cells. Genetic analysis of the HA, NS1, NA, and PB1 gene segments by Sanger sequencing revealed the presence of specific mutations and a repertoire of viral quasispecies following serial passaging. Most quasispecies were also found in PB1, which exhibited consistently high transversion-to-transition ratios in all five MDCK-SIAT1 passages. Most notably, passage 5 virus harbored the D457G substitution in the HA2 subunit, while passage 3 virus acquired K53Q and Q69H mutations in PB1-F2. An A971 variant leading to a non-synonymous R316Q substitution in PB1 was also identified in MDCK-SIAT1 passages 2 and 4. With an increasing number of passages, the proportion of D457G mutations progressively increased and was associated with larger virus plaque sizes. However, microneutralization assays revealed no significant differences in the neutralizing antibody profiles of human-influenza-immune serum samples against pre-passaged virus and passage 5 virus. In contrast, viable virus was only detected in passage 1 of CEF cells, which gave rise to multiple viral RNA quasispecies. Our findings highlight that serial passaging is able to drive differential adaptation of H3N2 influenza in different host species and may alter viral virulence. More studies are warranted to elucidate the complex relationships between H3N2 virus evolution, viral virulence changes, and low vaccine efficacy.

Hepatitis C virus (HCV) infection remains a serious public health burden around the world. So far there is no effective vaccine against this virus. Neutralizing antibody (NAb) responses to the epitopes within HCV E1 and E2 proteins are related to the resolution of hepatitis C infection. E. coli heat-labile enterotoxin B subunit (LTB) has been described as potent immunity adjuvants. In this study, we constructed recombinant pET vectors: pET-R9-Bp (B cell polyepitopes) expressing 7 epitopes from HCV E1 and E2 proteins including R9 (E2384-411aa)-Bp (E1313-327aa-E2396-424aa-E2436-447aa-E2523-540aa-E2610-627aa-E2631-648aa) and pET-LTB-R9-Bp expressing LTB adjuvant in combination with R9-Bp. Recombinant proteins R9-Bp and LTB-R9-Bp were expressed successfully in E. coli and purified by the Ni-NTA column. Both R9-Bp and LTB-R9-Bp in BALB/c mice induced robust humoral immune response in the context of intraperitoneal or intramuscular immunization but not oral immunization. Intraperitoneal administration of LTB-R9-Bp induced a higher antibody titer (peak titer: 1:341000) than that of R9-Bp (peak titer: 1:85000) after the second boost (P = 0.0036 or 0.0002). However, comparable antibody peak titers were elicited for both R9-Bp and LTB-R9-Bp in intramuscular immunization albeit with significant difference (P = 0.0032) a week after the second boost. In addition, both R9-Bp and LTB-R9-Bp induced the secretion of cytokines including IFN-γ and IL-4 at similar levels. anti-sera induced by both R9-Bp and LTB-R9-Bp recognized native HCV E1 and E2 proteins. Moreover, these HCV-specific antisera inhibited significantly the entry of HCV (P < 0.0001). Taken together, these findings showed that E. coli-based both R9-Bp and LTB-R9-Bp could become promising HCV vaccines.