Danh Cong Lai

DNA-based vaccines have been developed and tested against a range of infectious agents, including influenza A viruses (IAV). Bacterial plasmid DNA is widely used as a vector for delivering genes encoding vaccine immunogens. However, the inclusion of antibiotic resistance genes in conventional plasmid constructs raises safety concerns. Linear DNA expression cassettes produced via PCR that lack antibiotic resistance genes, and bacterial sequences can serve as a safer alternative vector. In this study, we employed a mouse model of IAV to evaluate the protective efficacy of a lipid nanoparticle (LNP)-encapsulated linear DNA expression cassette containing the hemagglutinin gene of IAV H1N1 produced by PCR amplification. For comparison, a circular plasmid DNA containing the same gene was also constructed. The LNP-encapsulated DNA cassette (LNP-L) exhibited similar physicochemical characteristics, such as particle size, polydispersity index, zeta potential, and encapsulation efficiency, compared to the LNP-encapsulated circular plasmid DNA (LNP-P). However, LNP-L produced significantly lower transgene expression than LNP-P in cell culture. Mice immunized with LNP-L developed a delayed onset of antibody response compared to those immunized with LNP-P, but the magnitudes of both humoral and cell-mediated immune responses were comparable between the two groups after booster immunization. Moreover, mice immunized with LNP-L exhibited both Th1 and Th2 responses, similar to those immunized with LNP-P. Importantly, mice immunized with either LNP-L or LNP-P were fully protected upon challenge with a lethal dose of IAV H1N1. These findings suggest that linear DNA expression cassettes encapsulated in LNPs represent a promising strategy for vaccine development. IMPORTANCE: Lipid nanoparticle (LNP)-encapsulated DNA vaccines represent a promising platform for the rapid development of vaccines against infectious pathogens. However, the inclusion of an antibiotic-resistance gene in plasmid DNA vectors used to deliver vaccine immunogens raises potential safety concerns. This study explores the feasibility of using a linear expression cassette produced via PCR amplification as an alternative system for delivering vaccine immunogens. Compared with conventional plasmid DNA, the linear expression cassette contains only essential elements, including a promoter, the gene encoding the vaccine immunogen, and a polyadenylation signal, and can be produced in a cell-free system. Using a mouse model for influenza A virus, we demonstrate that an LNP-encapsulated linear DNA cassette induces humoral and cell-mediated immune responses and protects immunized mice against a lethal influenza virus challenge. Thus, the LNP-encapsulated linear expression cassette may provide a safer approach for rapid responses to infectious diseases in animals.

Maternally derived antibodies (MDAs) play a vital role in protecting neonates from infectious diseases, but their presence at the time of vaccination can interfere with vaccine-induced immune responses, thereby reducing vaccine effectiveness. MDA interference has been documented in pigs vaccinated with whole-inactivated virus (WIV) vaccines against swine influenza A virus (IAV). This study evaluated the efficacy of a lipid nanoparticle-encapsulated DNA (LNP-DNA) vaccine against swine IAV in the presence and absence of MDAs, comparing it to a WIV vaccine. In MDA-negative piglets, both the LNP-DNA and WIV vaccines induced strong immune responses and effectively prevented the vaccinated animals from being infected with the homologous IAV strain. However, in MDA-positive piglets, the WIV vaccine failed to trigger significant antibody or T-cell responses and offered no protection against viral shedding or lung damage. In contrast, the LNP-DNA vaccine elicited stronger immune responses in MDA-positive pigs, reduced nasal viral shedding, and prevented lung lesions. These findings demonstrate that the LNP-DNA vaccine overcomes MDA interference, making it a promising strategy for enhancing vaccine efficacy in neonatal animals with maternal antibodies. IMPORTANCE: Maternally derived antibody (MDA) interference is a major obstacle to developing effective vaccines for neonates. In pigs, MDAs significantly impair immune responses to a whole-inactivated virus vaccine. Here, we show that vaccination with a lipid nanoparticle (LNP)-encapsulated DNA vaccine can partially overcome MDA interference. These findings underscore the potential of the LNP-DNA vaccine as a viable strategy for effectively immunizing MDA-positive populations. Additionally, LNP-DNA vaccination in young pigs provides a valuable model for exploring the immunological mechanisms behind MDA-mediated suppression of vaccine-induced immunity.

Background and Aim: Neonatal piglet diarrhea (NPD) remains a significant challenge in the swine industry, contributing to elevated pre-weaning mortality, reduced productivity, and increased economic losses. In Vietnam, despite the growing importance of commercial pig production, comprehensive studies investigating the epidemiology of NPD and associated bacterial pathogens are lacking. This study aimed to assess the prevalence of Escherichia coli, Clostridium perfringens, and Clostridioides difficile and to identify risk factors contributing to NPD on Vietnamese sow farms. Materials and Methods: A cross-sectional study was conducted on 40 commercial sow farms across North, Central, and South Vietnam between August and December 2023. Structured questionnaires captured data on farm characteristics, management practices, and health interventions. Fecal samples from symptomatic piglets aged 1–14 days were pooled and analyzed using multiplex quantitative polymerase chain reaction to detect virulence genes of E. coli (F4, F5, F6, LT), C. perfringens (alpha, beta, and epsilon toxins), and C. difficile (toxins A and B). Logistic and ordinal regression models were applied to assess associations between risk factors and pathogen prevalence. Results: All farms tested positive for at least one pathogen. C. perfringens was the most prevalent (97.37%), followed by E. coli (46.49%) and C. difficile (39.47%). Co-infections involving multiple pathogens were common (64.91%), with C. perfringens consistently present in all mixed infections. Key virulence genes detected included LT (35.96%), alpha toxin (95.61%), and toxin A (20.17%). Significant risk factors included farm type, region, weaning performance, and peripartum antibiotic administration route. Notably, farms using mixed-feed antibiotics exhibited higher E. coli prevalence. Larger farms and those practicing early piglet relocation also showed increased pathogen diversity. Conclusion: This study presents the first national-scale assessment of bacterial pathogens in NPD across Vietnamese sow farms. The findings highlight the high burden of toxin-producing bacteria, frequent co-infections, and multiple farm-level risk factors. Interventions such as targeted vaccination, optimized antibiotic use, improved weaning practices, and enhanced regional surveillance are essential for mitigating NPD impacts and improving piglet health outcomes in Vietnam. Keywords: bacterial toxins, Clostridioides difficile, Clostridium perfringens, Escherichia coli, farm management, multiplex quantitative polymerase chain reaction, neonatal piglet diarrhea, Vietnam, risk factors.

In 2009, a novel swine-origin H1N1 virus emerged, causing a pandemic. The virus, known as H1N1pdm09, quickly displaced the circulating H1 lineage and became the dominant seasonal influenza A virus subtype infecting humans. Human-to-swine spillovers of the H1N1pdm09 have occurred frequently, and each occurrence has led to sustained transmission of the human-origin H1N1pdm09 within swine populations. In the present study, we developed a lipid nanoparticle-based DNA vaccine (LNP-DNA) containing the hemagglutinin gene of a swine-origin H1N1pdm09. In pigs, this LNP-DNA vaccine induced a robust antibody response after a single intramuscular immunization and protected the pigs against challenge infection with the homologous swine-origin H1N1pdm09 virus. In a mouse model, the LNP-DNA vaccine induced antibody and T-cell responses and protected mice against lethal challenge with a mouse-adapted human-origin H1N1pdm09 virus. These findings demonstrate the potential of the LNP-DNA vaccine to protect against both swine- and human-origin H1N1pdm09 viruses. IMPORTANCE: Swine influenza A virus (IAV) is widespread and causes significant economic losses to the swine industry. Moreover, bidirectional transmission of IAV between swine and humans commonly occurs. Once introduced into the swine population, human-origin IAV often reassorts with endemic swine IAV, resulting in reassortant viruses. Thus, it is imperative to develop a vaccine that is not only effective against IAV strains endemic in swine but also capable of preventing the spillover of human-origin IAV. In this study, we developed a lipid nanoparticle-encapsulated DNA plasmid vaccine (LNP-DNA) that demonstrates efficacy against both swine- and human-origin H1N1 viruses. The LNP-DNA vaccines are non-infectious and non-viable, meeting the criteria to serve as a vaccine platform for rapidly updating vaccines. Collectively, this LNP-DNA vaccine approach holds great potential for alleviating the impact of IAV on the swine industry and preventing the emergence of reassortant IAV strains.

This study aimed to investigate the co-infection and genetic characteristics of Porcine circoviruses in PMWS-affected pigs in five commercial farrow-to-finish swine farms in Vietnam. By the end of 2022, the percentage of PMWS-affected pigs in these farms has increased significantly compared to previous years. The lymph node samples from ten PMWS typical cases were randomly collected to test for the presence of PRRSV, PCV2, PCV3 and PCV4. While PRRSV and PCV4 were not found in these cases, 10 and 3 out of 10 samples were positive for PCV2 and PCV3, respectively. Three farms in the study showed the co-infection of PCV2 and PCV3 in affected pigs. Besides, all PCV-positive samples were sequenced to evaluate genetic characterization of PCVs in PMWS-affected cases. Phylogenetic analysis showed that all PCV3 strains in the study were clustered into PCV3b genotype. 8 out of 10 PCV2 strains belonged to PCV2d genotype while the remaining two strains belonged to PCV2b genotypes. Two farms had co-circulation of PCV2b and PCV2d genotypes in two different age groups of pigs, which is reported for the first time in Vietnam. Several amino acid substitutions were identified in important antigenic regions in the capsid protein of the PCV2 field strains compared to vaccine strains. Taken together, the results showed the high co-prevalence of PCV3 and PCV2, and the wide genetic diversity of PCV2 field and vaccine strains may be the cause of the increased PMWS situation in these pig farms.

The Influenza A virus of swine (IAV-S) is highly prevalent and causes significant economic losses to swine producers. Due to the highly variable and rapidly evolving nature of the virus, it is critical to develop a safe and versatile vaccine platform that allows for frequent updates of the vaccine immunogens to cope with the emergence of new viral strains. The main objective of this study was to assess the feasibility of using lipid nanoparticles (LNPs) as nanocarriers for delivering DNA plasmid encoding the viral hemagglutinin (HA) gene in pigs. The intramuscular administration of a single dose of the LNP-DNA vaccines resulted in robust systemic and mucosal responses in pigs. Importantly, the vaccinated pigs were fully protected against challenge infection with the homologous IAV-S strain, with only 1 out of 12 vaccinated pigs shedding a low amount of viral genomic RNA in its nasal cavity. No gross or microscopic lesions were observed in the lungs of the vaccinated pigs at necropsy. Thus, the LNP-DNA vaccines are highly effective in protecting pigs against the homologous IAV-S strain and can serve as a promising platform for the rapid development of IAV-S vaccines.

Porcine epidemic diarrhea (PED) virus (PEDV) is a highly contagious virus. PED was first identified in 2008 and has greatly affected the Vietnamese pig production economy. The aim of this study was to investigate the epidemiological and genetic characteristics of PEDV in piglet herds in the Mekong Delta, Vietnam. Diarrheal stool and intestinal samples from 2262 piglets from 191 herds in five provinces were collected to test for the presence of PEDV. Ten PEDV strains were randomly selected for sequencing, and four genes encoding PEDV structural proteins were analyzed. The rates of herds and samples positive for PEDV were 27.23% and 27.72%, respectively. In positive herds, the morbidity and mortality of PEDV-positive piglets were 97.97% and 79.06%, respectively, with most of the infected piglets under 7 days of age. Phylogenetic analysis showed that the 10 PEDV strains from this study clustered with genotype G2 strains from Vietnam and neighboring countries. Many amino acid substitutions were identified in important antigenic regions in the spike protein of the 10 strains when compared to four PEDV vaccine strains. This study provides novel insights into the epidemiology and genetic diversity of circulating PEDV strains, which could facilitate the development of an appropriate and proactive strategy for controlling PED.

Porcine circovirus type 3 (PCV3) was first detected in 2016 and has been reported in many pig-producing countries around the world, including Vietnam. PCV3 has been found in complex cases with multiple clinical syndromes in swine. In this study, we investigated the genetic diversity of PCV3 strains circulating in Vietnam. A total of 249 samples were collected from swine farms located in eight provinces of Vietnam, and 11.65% (29/249) of these samples were found to contain PCV3. The ORF2 genes from the 29 PCV3-positive samples were amplified, purified, and sequenced. Phylogenetic analysis showed that 23 of these strains belonged to the PCV3b subtype, while the remaining six strains belonged to subtype c and subtype a (a-1 and a-2). Analysis of the ORF2 genes indicated that the 29 PCV3 strains had high sequence identity (96.90-100% at the genomic level and 96.19-100% at the amino acid level). Fifteen amino acid substitutions were found in predicted B-cell epitopes in the capsid proteins of the Vietnamese PCV3 strains.