Impact of genetic diversity of European-type porcine reproductive and respiratory syndrome virus strains on vaccine efficacy

The aim of this study was to find out how efficiently pigs that are vaccinated with an attenuated porcine reproductive and respiratory syndrome virus (PRRSV) vaccine based on a virus from the Lelystad cluster are protected against a European wild-type strain from the same or another genetic cluster. Two experiments were performed. In each experiment, 5-week-old PRRSV-seronegative pigs were vaccinated intramuscularly with 10(4.5) TCID50 of a commercial vaccine based on a European virus strain from the Lelystad cluster. Non-vaccinated pigs were included as controls. At 5, 9, 15, 20, 28, 35 and 42 days post vaccination (PV), broncho-alveolar lavage (BAL) fluids and blood were collected to determine vaccine virus quantities. Forty-nine days PV, pigs were challenged intranasally with 10(6.0) TCID50 of a European wild-type strain, belonging either to the Lelystad cluster (98% nucleotide identity in ORF5 with vaccine strain) (experiment A) or to an Italian cluster (84% nucleotide identity in ORF5 with vaccine strain) (experiment B). At 5, 9, 15, 20 and 27 days post challenge (PC), BAL fluids and blood were collected to determine virus quantities. Vaccine virus was first detected in BAL fluids and blood at 5 days PV and reached highest quantities between 9 and 15 days PV. One pig was positive in its BAL fluid until 42 days PV. After challenge, virus was isolated from BAL fluids and blood of all non-vaccinated control pigs. All vaccinated pigs challenged with the Lelystad strain remained negative for virus, while virus was present in BAL fluids and blood of all vaccinated pigs after challenge with the Italian strain. Mean virus titres of the vaccinated pigs challenged with the Italian strain were significantly lower than those of the non-vaccinated control pigs (P <0.05) at 9, 15 and 20 days PC. Thus, the genetic diversity within European-type PRRSV may affect the efficacy of the current European-type vaccines.     

Genetic diversity and phylogenetic analysis of porcine reproductive and respiratory syndrome virus isolates in East China

Porcine reproductive and respiratory syndrome virus (PRRSV) has been reported to have evolved at a high evolutionary rate and the extensive genetic variation. In this study, 44 PRRSV positive cases obtained from different provinces of China were sequenced and analyzed. Comparative analysis of partial isolates based on nsp2 sequences revealed that highly pathogenic PRRSV were the dominant viruses in China from 2008 to 2010 and some novel strains with an extra deletion of 19 aa. Phylogenetic analysis based on the GP5 genes showed that the PRRSV isolates from 1996 to 2010 had a great variation and the North American genotype was further divided into six subgenotypes. No apparent relationship between the heterogeneity and the geographic origin of isolates was observed. The 44 isolates and 29 representative strains were divided into six subgenotypes. Further analysis of the GP5 protein suggested that these strains of subgenotypes I, II and III exhibited variations in the primary neutralizing epitope and almost all isolates of subgenotypes II and III had more N-linked glycosylation sites. In addition, some mutations which could mirror the viral evolution and adaptation were also observed in this study. All these results might be useful to study the genetic variation and genetic relatedness among PRRSV strains in China.     

Vaccine efficacy of porcine reproductive and respiratory syndrome virus chimeras

The vaccine efficacy of six PRRSV Type 2 infectious clones, including five chimeras and a strain-specific deletion mutant, were examined using a respiratory challenge model in growing swine. The chimeras were constructed from different combinations of a licensed modified live vaccine (Ingelvac^® PRRS MLV) and a virulent field isolate (wt MN184) which differ by 14.3% on a nucleotide basis, while the deletion mutant tested had a broad deletion in the nsp2 region of strain MN184. The appearance of antibodies and virus characterization revealed regions of the genome that could influence PRRSV replication in vivo. Swine growth, clinical signs and lung lesions were also monitored. Average daily weight gain was negatively and directly impacted by some vaccines, and after challenge, vaccination with different constructs led to variable weight gain. We determined that 3 of the tested chimeras, including two previously published chimeras [1] and one in which strain MN184 ORF5-6 was placed on the background of Ingelvac^® PRRS MLV were able to prevent lung consolidation to a similar extent as traditionally prepared cell-passaged attenuated vaccines. The study suggested that only specific chimeras can attenuate clinical signs in swine and that attenuation cannot be directly linked to primary virus replication. Additionally, the strain MN184 deletion mutant was not found to have been sufficiently attenuated nor efficacious against heterologous challenge with strain JA-142.     

Evaluating an automated clustering approach in a perspective of ongoing surveillance of porcine reproductive and respiratory syndrome virus (PRRSV) field strains

Porcine reproductive and respiratory syndrome virus (PRRSV) has a major economic impact on the swine industry. The important genetic diversity needs to be considered for disease management. In this regard, information on the circulating endemic strains and their dispersal patterns through ongoing surveillance is beneficial. The objective of this project was to classify Quebec PRRSV ORF5 sequences in genetic clusters and evaluate stability of clustering results over a three-year period using an in-house automated clustering system. Phylogeny based on maximum likelihood (ML) was first inferred on 3661 sequences collected in 1998–2013 (Run 1). Then, sequences collected between January 2014 and September 2016 were sequentially added into 11 consecutive runs, each one covering a three-month period. For each run, detection of clusters, which were defined as groups of ≥15 sequences having a≥70% rapid bootstrap support (RBS) value, was automated in Python. Cluster stability was described for each cluster and run based on the number of sequences, RBS value, maximum pairwise distance and agreement in sequence assignment to a specific cluster. First and last run identified 29 and 33 clusters, respectively. In the last run, about 77% of the sequences were classified by the system. Most clusters were stable through time, with sequences attributed to one cluster in Run 1 staying in the same cluster for the 11 remaining runs. However, some initial groups were further subdivided into subgroups with time, which is important for monitoring since one specific wild-type cluster increased from 0% in 2007 to 45% of all sequences in 2016. This automated classification system will be integrated into ongoing surveillance activities, to facilitate communication and decision-making for stakeholders of the swine industry.     

Analysis of synonymous codon usage in porcine reproductive and respiratory syndrome virus

In this study, we calculated the relative synonymous codon usage (RSCU) values and codon usage bias (CUB) values to implement a comparative analysis of codon usage pattern of open reading frames (ORFs) which belong to the two main genotypes of porcine reproductive and respiratory syndrome virus (PRRSV). By analysis of synonymous codon usage values in each ORF of PRRSV, the optimal codons for most amino acids were all C or G-ended codons except GAU for Asp, CAU for His, UUU for Phe and CCU for Pro. The synonymous codon usage patterns in different ORFs of PRRSV were different and genetically conserved. Among them, ORF1a, ORF4, ORF5 and ORF7 could cluster these strains into the two main serotypes (EU and US). Due to mutational pressure, compositional constraint played an important role in shaping the synonymous codon usage pattern in different ORFs, and the synonymous codon usage diversity in ORFs was correlated with gene function. The degree of CUB for some particular amino acids under strong selection pressure probably served as a potential genetic marker for each ORF in PRRSV. However, gene length and translational selection in nature had no effect on the synonymous codon usage pattern in PRRSV. These conclusions could not only offer an insight into the synonymous codon usage pattern and differentiation of gene function, but also assist in understanding the discrepancy of evolution among ORFs in PRRSV.     

Challenges for porcine reproductive and respiratory syndrome virus (PRRSV) vaccinology

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be a threat for the pig industry. Vaccines have been developed, but these failed to provide sustainable disease control, in particular against genetically unrelated strains. Here we give an overview of current knowledge and gaps in our knowledge that may be relevant for the development of a future generation of more effective vaccines. PRRSV replicates in cells of the monocyte/macrophage lineage, induces apoptosis and necrosis, interferes with the induction of a proinflammatory response, only slowly induces a specific antiviral response, and may cause persistent infections. The virus appears to use several evasion strategies to circumvent both innate and acquired immunity, including interference with antigen presentation, antibody-mediated enhancement, reduced cell surface expression of viral proteins, and shielding of neutralizing epitopes. In particular the downregulation of type I interferon-α production appears to interfere with the induction of acquired immunity. Current vaccines are ineffective because they suffer both from the immune evasion strategies of the virus and the antigenic heterogeneity of field strains. Future vaccines therefore must “uncouple” the immune evasion and apoptogenic/necrotic properties of the virus from its immunogenic properties, and they should induce a broad immune response covering the plasticity of its major antigenic sites. Alternatively, the composition of the vaccine should be changed regularly to reflect presently and locally circulating strains. Preferably new vaccines should also allow discriminating infected from vaccinated pigs to support a virus elimination strategy. Challenges in vaccine development are the incompletely known mechanisms of immune evasion and immunity, lack of knowledge of viral sequences that are responsible for the pathogenic and immunosuppressive properties of the virus, lack of knowledge of the forces that drive antigenic heterogeneity and its consequences for immunogenicity, and a viral genome that is relatively intolerant for subtle changes at functional sites.     

Cross reactivity of immune responses to porcine reproductive and respiratory syndrome virus infection

Because porcine reproductive and respiratory syndrome virus (PRRSV) exhibits extensive genetic variation among field isolates, characterizing the extent of cross reactivity of immune responses, and most importantly cell-mediated immunity (CMI), could help in the development of broadly cross-protective vaccines. We infected 12 PRRSV-naïve pigs with PRRSV strain FL12 and determined the number of interferon (IFN)-γ secreting cells (SC) by ELISpot assay using ten type 2 and one type 1 PRRSV isolates as recall antigens. The number of IFN-γ SC was extremely variable among animals, and with exceptions, late to appear. Cross reactivity of IFN-γ SC among type 2 isolates was broad, and we found no evidence of an association between increased genetic distance among isolates and the intensity of the CMI response. Comparable to IFN-γ SC, total antibodies evaluated by indirect immunofluorescence assay (IFA) were cross reactive, however, neutralizing antibody titers could only be detected against the strain used for infection. Finally, we observed a moderate association between homologous IFN-γ SC and neutralizing antibodies.     

Inhibition of porcine reproductive and respiratory syndrome virus by Cecropin D in vitro

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to cause substantial economic losses to the pig industry worldwide. Although vaccines are commercially available for the control of PRRSV infection, no vaccination regimen has been proved sustained success in terms of generating a protective immune response. Therefore, the development of novel antivirals is urgently needed. Antimicrobial peptides display broad-spectrum antimicrobial activities against bacteria, fungi, and viruses and play an important role in host innate immune response. Here, we tested whether Cecropin D (CD) could inhibit PRRSV infection and replication in vitro. The inhibitory effect of CD occurred during viral attachment and the early period of viral entry into Marc-145 cells. CD also attenuated virus-induced apoptosis during the late phase of PRRSV infection and suppressed virus release in Marc-145 cells, which might contribute to the inhibition of PRRSV infection. Similar inhibitory effects on PRRSV infection were also found with CD treatment in porcine alveolar macrophages, the major target cell type of PRRSV infection in pigs in vivo. These findings suggest that CD has the potential to develop a new therapeutic agent against PRRSV infection.     

Positioning Quebec ORF5 sequences of porcine reproductive and respiratory syndrome virus (PRRSV) within Canada and worldwide diversity

Sequencing of ORF5 gene is widely used and considered essential for diagnostics and control of porcine reproductive and respiratory syndrome (PRRS) in Canada. The objective of this study was to position Quebec ORF5 sequences of PRRS virus within Canada and worldwide diversity. Overall, 76.8% of the 5204 sequences gathered from Quebec (n = 5031), Ontario (n = 151) and Manitoba (n = 18) were classified into one of 34 genetic clusters defined as groupings including ≥15 sequences and having ≥70% rapid bootstrap support value from a maximum likelihood (ML)-phylogeny. Following the addition of PRRSV 2 international reference dataset from Shi et al. (2010), the most predominant lineages in our dataset were wild-type 1 and vaccine-like 5.1 (MLV) and 8.9 (ATP). No strains or only a very few (1 or 2) were assigned to lineages 1.3–1.5, 3, 4, 5.2, 6, 7 or 9. Most wild-type clusters (97%) detected in a dataset from Canada did not include any sequence from the international reference dataset. It might reflect recent subpopulations that were absent at the time of Shi's publication. As an example, cluster #25 first appeared in 2007, but since then had expanded considerably and is now the most prevalent wild-type cluster found in Quebec. A total of 117 RFLP patterns were identified and those were poorly correlated with genetic clusters based on phylogeny. Factors modulating PRRSV diversity such as pig movement that occurred within and between provinces should be further investigated in a perspective of disease control.     

Development of a broadly protective modified-live virus vaccine candidate against porcine reproductive and respiratory syndrome virus

Modified-live virus (MLV) vaccines are widely used to protect pigs against porcine reproductive and respiratory syndrome virus (PRRSV). However, current MLV vaccines do not confer adequate levels of heterologous protection, presumably due to the substantial genetic diversity of PRRSV isolates circulating in the field. To overcome this genetic variation challenge, we recently generated a synthetic PRRSV strain containing a consensus genomic sequence of PRRSV-2. We demonstrated that our synthetic PRRSV strain confers unprecedented levels of heterologous protection. However, the synthetic PRRSV strain at passage 1 (hereafter designated CON-P1) is highly virulent and therefore, is not suitable to be used as a vaccine in pigs. In the present study, we attenuated CON-P1 by continuously passaging the virus in MARC-145 cells, a non-natural host cell line. Using a young pig model, we demonstrated that the synthetic virus at passages 90 and 122 (designated as CON-P90 and CON-P122, respectively) were fully attenuated, as evidenced by the significantly reduced viral loads in serum and tissues and the absence of lung lesion in the infected pigs. Most importantly, CON-P90 confers similar levels of heterologous protection as its parental strain CON-P1. Taken together, the results indicate that CON-P90 is an excellent candidate for the formulation of next generation of PRRSV MLV vaccines with improved levels of heterologous protection.     

Live porcine reproductive and respiratory syndrome virus vaccines: Current status and future direction

Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) was reported in the late 1980s. PRRS still is a huge economic concern to the global pig industry with a current annual loss estimated at one billion US dollars in North America alone. It has been 20 years since the first modified live-attenuated PRRSV vaccine (PRRSV-MLV) became commercially available. PRRSV-MLVs provide homologous protection and help in reducing shedding of heterologous viruses, but they do not completely protect pigs against heterologous field strains. There have been many advances in understanding the biology and ecology of PRRSV; however, the complexities of virus-host interaction and PRRSV vaccinology are not yet completely understood leaving a significant gap for improving breadth of immunity against diverse PRRS isolates. This review provides insights on immunization efforts using infectious PRRSV-based vaccines since the 1990s, beginning with live PRRSV immunization, development and commercialization of PRRSV-MLV, and strategies to overcome the deficiencies of PRRSV-MLV through use of replicating viral vectors expressing multiple PRRSV membrane proteins. Finally, powerful reverse genetics systems (infectious cDNA clones) generated from more than 20 PRRSV isolates of both genotypes 1 and 2 viruses have provided a great resource for exploring many innovative strategies to improve the safety and cross-protective efficacy of live PRRSV vaccines. Examples include vaccines with diminished ability to down-regulate the immune system, positive and negative marker vaccines, multivalent vaccines incorporating antigens from other porcine pathogens, vaccines that carry their own cytokine adjuvants, and chimeric vaccine viruses with the potential for broad cross-protection against heterologous strains. To combat this devastating pig disease in the future, evaluation and commercialization of such improved live PRRSV vaccines is a shared goal among PRRSV researchers, pork producers and biologics companies.     

Maternally-derived neutralizing antibodies reduce vaccine efficacy against porcine reproductive and respiratory syndrome virus infection

Modified live virus (MLV) vaccines are commonly used to reduce the impact of porcine reproductive and respiratory syndrome (PRRS) but limited efficacy is achieved in field conditions. Here, we evaluated the impact of maternally-derived neutralizing antibodies (MDNAs) on vaccine efficacy after PRRS virus (PRRSV) challenge. Piglets with low (A−) or high (A+) MDNA levels derived from a commercial pig herd were moved to experimental facilities to be vaccinated (V+) or not (V−) with a PRRSV-1 MLV vaccine at 3 weeks of age (woa). Because of unexpectedly low vaccine detection in A−V+ piglets post-vaccination (pv), all V+ piglets received a second vaccination at 4 woa. Five weeks (W5) pv, piglets were inoculated with a PRRSV-1 field strain to evaluate vaccine protection, and were mingled 24 h later with non-inoculated piglets of similar immune status to assess viral transmission. Vaccine strain was detected at W2 pv in 69% and 6% of A−V+ and A+V+ piglets, and at W5 pv in 50% and 25% of A−V+ and A+V+ piglets, respectively. At W5 pv, 94% of A−V+ and 44% of A+V+ piglets seroconverted, with a significant IFNg response induction in the A−V+ group only. After challenge, compared to the V− inoculated group, viremia was 100-fold lower at 10 days post-infection in A−V+ whereas viremia was not significantly reduced in A+V+ piglets. A lower transmission rate was estimated for the A−V+ group: 0.15 [0.07–0.29] versus 0.44 [0.18–1.76] and 0.32 [0.14–0.68] for the A+V+ and V− groups, respectively. Investigations about the low vaccine strain detection after the first vaccination suggested a relationship between IFNa levels and vaccine strain detection in A−V+ piglets. We showed that MDNAs impair vaccine efficacy against PRRSV both in inoculated and contact piglets, probably by reducing vaccine replication. IFNa may also interfere with PRRSV vaccination. These new data could help improving vaccination protocols.     

Enhancing neutralizing antibody production by an interferon-inducing porcine reproductive and respiratory syndrome virus strain

Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) continues to cause substantial economic losses to the global swine industry. PRRSV appears to inhibit synthesis of type I interferons (IFNs), such as IFN-α and -β, which are critical for the innate immunity and play an important role in the modulation of adaptive immunity. An atypical PRRSV strain, A2MC2, is able to induce type I IFNs in vitro. In this study, A2MC2 induction of neutralizing antibodies in vivo was compared with the Ingelvac PRRS modified live virus (MLV) vaccine strain and VR-2385 (a moderate virulent strain). Three-week-old pigs were exposed to these PRRSV strains via intranasal or intramuscular routes to also account for a possible effect of inoculation routes. The interferon-inducing A2MC2 resulted in earlier onset and significantly higher levels of PRRSV neutralizing antibodies than the MLV. In addition, the A2MC2-induced neutralizing antibodies were capable of neutralizing VR-2385, a heterologous strain. The pigs exposed via intranasal route had higher titers of neutralizing antibodies than those injected via intramuscular route. Macroscopic and microscopic lung lesions 14 days post-exposure indicated that A2MC2 had similar virulence in vivo as VR-2385. Pulmonary alveolar macrophages (PAMs) collected during the necropsy 14 days post-exposure in the A2MC2 group had higher level expression of IFN-γ than the MLV group. These results indicate that A2MC2 can be further explored for development of an improved vaccine against PRRS.     

Preparation and heat resistance study of porcine reproductive and respiratory syndrome virus sugar glass vaccine

To improve the preservation period without cold-chain of the live attenuated vaccine of porcine reproductive and respiratory syndrome (PRRS), a set of thermostable formulations composed of trehalose, tryptone and other protectants were dried by vacuum foam drying (VFD) along with PRRSV solutions. In the 37 °C and 45 °C resistance ageing test, the dried foam vaccine showed significant thermostability, and the virus titer lost 0.8 Log₁₀ at 37 °C for 4 months, 1.0 Log₁₀ at 45 °C for 25 days. Furthermore, the foam vaccine could be stored at 25 °C for at least one year. Besides, the vaccine preserved in 37 °C, 25 °C and 4 °C for 3 months were inoculated on 20-days old piglet, and the serum titer was monitoring by ELISA kit. Inoculated two weeks later, the ELISA titer were all qualified and had the similar level compared to the commercial vaccines of the lyophilization dosage.     

Chlorine dioxide inhibits the replication of porcine reproductive and respiratory syndrome virus by blocking viral attachment

Porcine reproductive and respiratory syndrome virus (PRRSV) causes a great economic loss to the swine industry globally. Current prevention and treatment measures are not effective to control the outbreak and spread of porcine reproductive and respiratory syndrome (PRRS). In other words, new antiviral strategies are urgently needed. Chlorine dioxide (ClO₂) is regarded as a broad-spectrum disinfectant with strong inhibitory effects on microbes and parasites. The purpose of this study was to evaluate the inhibitory effects and underlying molecular mechanisms of ClO₂ against PRRSV infection in vitro. Here, we identified ClO₂ (the purity is 99%) could inhibit the infection and replication of PRRSV in both Marc-145 cells and porcine alveolar macrophages (PAMs). ClO₂ could block PRRSV binding to cells rather than internalization and release, suggesting that ClO₂ blocks the first stage of the virus life cycle. We also demonstrated that the inhibition exerted by ClO₂ was attributed to the degradation of PRRSV genome and proteins. Moreover, we confirmed that ClO₂ could decrease the expression of inflammatory cytokines induced by PRRSV. In summary, ClO₂ is an efficient agent and potently suppressed PRRSV infection in vitro.     

Whole genome characterization of a novel porcine reproductive and respiratory syndrome virus 1 isolate: Genetic evidence for recombination between Amervac vaccine and circulating strains in mainland China

Genotype 1 porcine reproductive and respiratory syndrome virus (PRRSV 1) have been continuously isolated in China in recent years. Complete genome sequences of these isolates are important to investigate the prevalence and evolution of Chinese PRRSV 1. Herein, we describe the isolation of a novel PRRSV 1 isolate, denominated HLJB1, in the Heilongjiang province of China. Complete genome sequencing of HLJB1 showed that it shares 90.66% and 58.21% nucleotide identities with PRRSV 1 and 2 prototypic strains Lelystad virus and ATCC VR-2332, respectively. HLJB1 has a unique 5-amino-acid insertion in nsp2, which has never been described in other PRRSV 1 isolates. Whole genome-based phylogenetic analysis revealed that all Chinese PRRSV 1 isolates are clustered in pan-European subtype 1 and can be divided into four subgroups. HLJB1 resides in the subgroup of BJEU06-1-like isolates but is also closely related to the Amervac-like isolates. Additionally, recombination analyses suggested that HLJB1 is a recombinant from the Amervac vaccine and the BJEU06-1 isolate. To our best knowledge, our results provide the first genetic evidence for recombination between Amervac vaccine and circulating strains. These findings are also beneficial for studying the origin and evolution of PRRSV 1 in China.     

Inhibition of porcine reproductive and respiratory syndrome virus by specific siRNA targeting Nsp9 gene

To screen siRNAs for effectively inhibiting the replication of porcine reproductive and respiratory syndrome virus (PRRSV). Four pairs of siRNA targeting Nsp9 gene of PRRSV and one non-efficient pair used as control were designed, synthesized and cloned into pSilencer4.1-CMV neo, designated as pSi-294, pSi-367, pSi-409, pSi-1488, pSi-Ctr. The recombinant plasmids were transfected into Marc-145 cells and infected with PRRSV 24 h post transfection. Subsequently, IFA, real-time PCR, TCID₅₀ and western blot were used for evaluating the inhibitory effect of the siRNA. IFA and western-blot results showed that pSi-294, pSi-1488 can effectively inhibit the expression of Nsp9 and M protein of PRRSV, real-time PCR result showed that the expression of Nsp9 gene were decreased from 86.56% to 93.66% compared to the negative control. siRNAs can be used as candidates for basic research of PRRSV.     

Peptide nanofiber hydrogel adjuvanted live virus vaccine enhances cross-protective immunity to porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) is prevalent in swine farms worldwide and is a major source of economic loss and animal suffering. Rapid genetic variation of PRRSV makes it difficult for current vaccines to confer protection against newly emerging strains. We recently demonstrated that a novel peptide nanofiber hydrogel (H9e) could act as a potent adjuvant for killed H1N1 vaccines. Therefore, the objective of this study was to evaluate H9e as an adjuvant for PRRSV modified live virus (MLV) vaccines. Pigs were vaccinated with Ingelvac PRRSV MLV with or without H9e adjuvant before being challenged with the VR-2332 (parental vaccine strain) or MN184A (genetically diverse strain) PRRSV. Pigs vaccinated with MLV + H9e had higher levels of circulating vaccine virus. More importantly, pigs vaccinated with MLV + H9e had improved protection against challenge by both PRRSV strains, as demonstrated by reduced challenge-induced viremia compared with pigs vaccinated with MLV alone. Pigs vaccinated with MLV + H9e had lower frequency of T-regulatory cells and IL-10 production but higher frequency of Th/memory cells and IFN-γ secretion than that in pigs vaccinated with MLV alone. Taken together, our studies suggest that the peptide nanofiber hydrogel H9e, when combined with the PRRSV MLV vaccine, can enhance vaccine efficacy against two different PRRSV strains by modulating both host humoral and cellular immune responses.