Kanjana Srisutthisamphan

Background: The continual emergence of antigenically drifted avian influenza viruses poses a persistent threat to global health and underscores the need for broadly protective approaches. Stem-directed monoclonal antibodies targeting conserved hemagglutinin (HA) epitopes are a promising strategy to address viral diversity. CR9114 is a broadly neutralizing antibody previously reported to recognize a conserved HA stem region across influenza A and B viruses. Methods: CR9114 was transiently expressed in Nicotiana benthamiana and characterized for protein integrity, assembly, and glycosylation. Binding to recombinant hemagglutinin was assessed, and neutralizing activity was evaluated against antigenically distinct avian influenza A viruses using in vitro neutralization assays. Results: Plant-produced CR9114 was correctly assembled as a human IgG1 κ antibody and displayed a high-mannose glycosylation profile. The antibody showed strong binding to recombinant H5 hemagglutinin (Kd = 0.15 µg/mL) and potently neutralized recent avian influenza isolates, namely A/Jiangsu/NJ210/2023 (H5N1; NT₅₀ = 1589) and A/Gansu/23277/2019 (H7N9; NT₅₀ = 177), demonstrating cross-subtype neutralization despite known glycan-associated constraints in Group 2 viruses. Conclusions: These findings demonstrate that N. benthamiana is a viable platform for the rapid production of functional broadly neutralizing anti-influenza antibodies. The preserved activity of plant-produced CR9114 against contemporary avian influenza strains supports its continued evaluation as a broadly protective therapeutic candidate and highlights the potential of plant molecular pharming approaches to contribute to pandemic preparedness.

Background/Objectives: African swine fever (ASF) is a devastating disease affecting the swine industry globally. Development of safe and effective vaccines is an urgent need. This study aimed to evaluate, caASFV001-MA52, a cell-adapted ASFV strain derived from serial passaging in MA-104 cells, as a promising live-attenuated vaccine (LAV) candidate against virulent ASFV infection. Methods: Seven-week-old, crossbred pigs were immunized with caASFV001-MA52 (at a dose of 10⁵ TCID₅₀) and subsequently challenged with a lethal dose of virulent ASFV. Vaccine efficacy was measured through clinical monitoring, immunological and virological analyses, and pathological assessments of tissue protection and viral load reduction. Safety was critically assessed, particularly regarding its profile in animals with concurrent endemic porcine infections, including PCV, PRRSV and S. suis. Results: caASFV001-MA52 exhibits partial protection (70–80%) against the lethal challenge. Vaccinated and challenged survivors exhibited reduced viral loads and significantly alleviated pathological lesions compared to controls. Safety evaluations revealed that the vaccine’s profile is susceptible to concurrent infection. Pigs co-infected with endemic porcine pathogens showed increased virulence and mortality following vaccination. Although vaccination temporarily reactivated latent viral infections (PCV2, PCV3, and PRRSV), most surviving pigs effectively controlled and eliminated these co-infections. Conclusions: The caASFV001-MA52 strain demonstrates promising immunogenicity and protection against lethal challenges, supporting its continued development as an LAV candidate. However, the observed safety concerns regarding concurrent infections emphasize the critical need for veterinary health surveillance during its future practical application.

Background: Middle East Respiratory Syndrome Coronavirus (MERS-CoV) causes severe respiratory illness in humans and currently lacks an approved vaccine. The Newcastle disease virus (NDV) vector is a well-established, safe, and effective platform for vaccine development. With recent advancements in stabilizing coronavirus spike proteins to enhance their antigenicity, this study aimed to determine whether modifications to the MERS-CoV spike protein could improve its presentation on NDV particles, allowing the resulting virus to be used as an inactivated vaccine. Methods: We codon-optimized the gene encoding the ectodomain of the MERS-CoV spike protein and incorporated modifications at the S1/S2 and S2’ cleavage sites, along with a proline substitution at residues V1060-L1061. This modified spike gene was inserted into the NDV genome to create the NDV-S_MERS virus. After purification and inactivation, the vaccine’s immunogenicity was assessed in mice. Results: Mice immunized with the inactivated NDV-S_MERS vaccine developed robust anti-spike IgGs, neutralizing antibodies, and cellular immune responses. The study demonstrated that modifications to the MERS-CoV spike protein were essential for its effective presentation on NDV particles. Additionally, the spike gene insert remained stable through five egg passages, confirming the vector’s stability. Conclusions: Engineering the MERS-CoV spike protein is crucial for its successful display on NDV particles. The strong immune responses elicited by the NDV-S_MERS vaccine in mice highlight that NDV is a promising, safe, and effective platform for MERS-CoV vaccination.

Subunit vaccines stand as a leading approach to expanding the current portfolio of vaccines to fight against COVID-19, seeking not only to lower costs but to achieve long-term immunity against variants of concern and have the main attributes that could overcome the limitations of the current vaccines. Herein a chimeric protein targeting S1 and S2 epitopes, called LTp50, was designed as a convenient approach to induce humoral responses against SARS-CoV-2. LTp50 was produced in recombinant Escherichia coli using a conventional pET vector, recovering the expected antigen in the insoluble fraction. LTp50 was purified by chromatography (purity > 90%). The solubilization and refolding stages helped to obtain a stable protein amenable for vaccine formulation. LTp50 was adsorbed onto alum, resulting in a stable formulation whose immunogenic properties were assessed in BALB/c mice. Significant humoral responses against the S protein (BA.5 variant) were detected in mice subjected to three subcutaneous doses (10 µg) of the LTp50/alum formulation. This study opens the path for the vaccine formulation optimization using additional adjuvants to advance in the development of a highly effective anti-COVID-19 vaccine directed against the antigenic regions of the S protein, which are less prone to mutations.

Coronaviruses isolated from bats and pangolins are closely related to SARS-CoV-2, the causative agent of COVID-19. These so-called sarbecoviruses are thought to pose an acute pandemic threat. As SARS-CoV-2 infection and vaccination have become more widespread, it is not known whether neutralizing antibodies to SARS-CoV-2 can cross-neutralize coronaviruses transmitted by bats or pangolins. In this study, we analyzed antibody-mediated neutralization with serum samples from COVID-19 patients (n = 31) and those immunized with inactivated SARS-CoV-2 vaccines (n = 20) against lentivirus-based pseudo-viruses carrying the spike derived from ancestral SARS-CoV-2, bat (RaTG13 or RshSTT182), or pangolin coronaviruses (PCoV-GD). While SARS-CoV-2, PCoV-GD, and RshSTT182 spikes could promote cell-cell fusion in VeroE6 cells, the RaTG13 spike did not. RaTG13, on the other hand, was able to induce cell-cell fusion in cells overexpressing ACE2. Dramatic differences in neutralization activity were observed, with the highest level observed for RaTG13, which was even significantly higher than SARS-CoV-2, PCoV-GD, and RshSTT182 pseudo-viruses. Interestingly, pseudo-viruses containing the chimeric protein in which the receptor-binding domain (RBD) of PCoV-GD spike was replaced by that of RaTG13 could be strongly neutralized, whereas those carrying RaTG13 with the RBD of PCoV-GD were significantly less neutralized. Because the high neutralizing activity against RaTG13 appears to correlate with its low affinity for binding to the human ACE2 receptor, our data presented here might shed light on how pre-existing immunity to SARS-CoV-2 might contribute to protection against related sarbecoviruses with potential spillover to the human host.

Virus-like particles (VLPs) are highly immunogenic and versatile subunit vaccines composed of multimeric viral proteins that mimic the whole virus but lack genetic material. Due to the lack of infectivity, VLPs are being developed as safe and effective vaccines against various infectious diseases. In this study, we generated a chimeric VLP-based COVID-19 vaccine stably produced by HEK293T cells. The chimeric VLPs contain the influenza virus A matrix (M1) proteins and the SARS-CoV-2 Wuhan strain spike (S) proteins with a deletion of the polybasic furin cleavage motif and a replacement of the transmembrane and cytoplasmic tail with that of the influenza virus hemagglutinin (HA). These resulting chimeric S-M1 VLPs, displaying S and M1, were observed to be enveloped particles that are heterogeneous in shape and size. The intramuscular vaccination of BALB/c mice in a prime-boost regimen elicited high titers of S-specific IgG and neutralizing antibodies. After immunization and a challenge with SARS-CoV-2 in K18-hACE2 mice, the S-M1 VLP vaccination resulted in a drastic reduction in viremia, as well as a decreased viral load in the lungs and improved survival rates compared to the control mice. Balanced Th1 and Th2 responses of activated S-specific T-cells were observed. Moderate degrees of inflammation and viral RNA in the lungs and brains were observed in the vaccinated group; however, brain lesion scores were less than in the PBS control. Overall, we demonstrate the immunogenicity of a chimeric VLP-based COVID-19 vaccine which confers strong protection against SARS-CoV-2 viremia in mice.

In response to the SARS-CoV-2 Delta variant, which partially escaped the vaccine-induced immunity provided by two doses of vaccination with CoronaVac (Sinovac), the National Vaccine Committee recommended the heterologous CoronaVac-ChAdOx1 (Oxford–AstraZeneca), a prime–boost vaccine regimen. This pilot study aimed to describe the immunogenicity and adverse events of the heterologous CoronaVac-ChAdOx1 regimen, in comparison with homologous CoronaVac, and homologous ChAdOx1. Between May and August 2021, we recruited a total of 354 participants from four vaccination groups: the CoronaVac-ChAdOx1 vaccinee (n = 155), the homologous CoronaVac vaccinee (n = 32), the homologous ChAdOx1 vaccinee (n = 47), and control group of COVID-19 patients (n = 120). Immunogenicity was evaluated by measuring the level of IgG antibodies against the receptor-binding domain (anti-SRBD) of the SARS-CoV-2 spike protein S1 subunit and the level of neutralizing antibodies (NAbs) against variants of concern (VOCs) using the plaque reduction neutralization test (PRNT) and pseudovirus neutralization test (pVNT). The safety profile was recorded by interviewing at the 1-month visit after vaccination. The anti-SRBD level after the second booster dose of the CoronaVac-ChAdOx1 group at 2 weeks was higher than 4 weeks. At 4 weeks after the second booster dose, the anti-SRBD level in the CoronaVac-ChAdOx1 group was significantly higher than either homologous CoronaVac, the homologous ChAdOx1 group, and Control group (p < 0.001). In the CoronaVac-ChAdOx1 group, the PRNT₅₀ level against the wild-type (434.5 BAU/mL) was the highest; followed by Alpha variant (80.4), Delta variant (67.4), and Beta variant (19.8). The PVNT₅₀ level was also found to be at its highest against the wild-type (432.1); followed by Delta variants (178.3), Alpha variants (163.9), and Beta variant (42.2), respectively. The AEs in the CoronaVac-ChAdOx1 group were well tolerated and generally unremarkable. The CoronaVac-ChAdOx1 heterologous regimen induced higher immunogenicity and a tolerable safety profile. In a situation when only CoronaVac-ChAdOx1 vaccines are available, they should be considered for use in responding to the Delta variant.

The use of virus-vectored platforms has increasingly gained attention in vaccine development as a means for delivering antigenic genes of interest into target hosts. Here, we describe a single-cycle influenza virus-based SARS-CoV-2 vaccine designated as scPR8-RBD-M2. The vaccine utilizes the chimeric gene encoding 2A peptide-based bicistronic protein cassette of the SARS-CoV-2 receptor-binding domain (RBD) and influenza matrix 2 (M2) protein. The C-terminus of the RBD was designed to link with the cytoplasmic domain of the influenza virus hemagglutinin (HA) to anchor the RBD on the surface of producing cells and virus envelope. The chimeric RBD-M2 gene was incorporated in place of the HA open-reading frame (ORF) between the 3′ and 5′ UTR of HA gene for the virus rescue in MDCK cells stably expressing HA. The virus was also constructed with the disrupted M2 ORF in segment seven to ensure that M2 from the RBD-M2 was utilized. The chimeric gene was intact and strongly expressed in infected cells upon several passages, suggesting that the antigen was stably maintained in the vaccine candidate. Mice inoculated with scPR8-RBD-M2 via two alternative prime-boost regimens (intranasal-intranasal or intranasal-intramuscular routes) elicited robust mucosal and systemic humoral immune responses and cell-mediated immunity. Notably, we demonstrated that immunized mouse sera exhibited neutralizing activity against pseudotyped viruses bearing SARS-CoV-2 spikes from various variants, albeit with varying potency. Our study warrants further development of a replication-deficient influenza virus as a promising SARS-CoV-2 vaccine candidate.

The Open Reading Frame 3 (ORF3), an accessory protein of porcine epidemic diarrhea virus (PEDV), has been shown to interact with a myriad of cellular proteins, among which include the IκB kinase β (IKBKB). Here, specific IKBKB domains responsible for ORF3–IKBKB interaction were identified. Dysregulation of NF-κB and Type I interferon (IFN) in the presence of ORF3 was also demonstrated. We showed that while ORF3 was capable of up-regulating IKBKB-meditated NF-κB promoter activity, it surprisingly down-regulated the activation of IKBKB-meditated IFN-β promoter and expression of IFN-β mRNA. When overexpressed, ORF3 could suppress Poly I:C mediated type I IFN production and induction. Finally, we demonstrated that IKBKB- and RIG-I-mediated type I IFN induction by ORF3 resulted in different outcomes. Our study is the first to demonstrate the potential and complex roles of ORF3 in the involvement of aberrant immune signaling as well as in the virus–host interaction.