Swine influenza virus (SIVs) infections cause a significant economic impact to the pork industry. Moreover, pigs may act as mixing vessel favoring genome reassortment of diverse influenza viruses. Such an example is the pandemic H1N1 (pH1N1) virus that appeared in 2009, harboring a combination of gene segments from avian, pig and human lineages, which rapidly reached pandemic proportions. In order to confront and prevent these possible emergences as well as antigenic drift phenomena, vaccination remains of vital importance. The present work aimed to evaluate a new DNA influenza vaccine based on distinct conserved HA-peptides fused with flagellin and applied together with Diluvac Forte as adjuvant using a needle-free device (IntraDermal Application of Liquids, IDAL®). Two experimental pig studies were performed to test DNA-vaccine efficacy against SIVs in pigs. In the first experiment, SIV-seronegative pigs were vaccinated with VC4-flagellin DNA and intranasally challenged with a pH1N1. In the second study, VC4-flagellin DNA vaccine was employed in SIV-seropositive animals and challenged intranasally with an H3N2 SIV-isolate. Both experiments demonstrated a reduction in the viral shedding after challenge, suggesting vaccine efficacy against both the H1 and H3 influenza virus subtypes. In addition, the results proved that maternally derived antibodies (MDA) did not constitute an obstacle to the vaccine approach used. Moreover, elevated titers in antibodies both against H1 and H3 proteins in serum and in bronchoalveolar lavage fluids (BALFs) was detected in the vaccinated animals along with a markedly increased mucosal IgA response. Additionally, vaccinated animals developed stronger neutralizing antibodies in BALFs and higher inhibiting hemagglutination titers in sera against both the pH1N1 and H3N2 influenza viruses compared to unvaccinated, challenged-pigs. It is proposed that the described DNA-vaccine formulation could potentially be used as a multivalent vaccine against SIV infections.

Influenza viruses represent a continuous threat to both animal and human health. The 2009 H1N1 A influenza pandemic highlighted the importance of a swine host in the adaptation of influenza viruses to humans. Nowadays, one of the most extended strategies used to control swine influenza viruses (SIVs) is the trivalent vaccine application, whose formulation contains the most frequently circulating SIV subtypes H1N1, H1N2, and H3N2. These vaccines do not provide full protection against the virus, allowing its replication, evolution, and adaptation. To better understand the main mechanisms that shape viral evolution, here, the SIV intra-host diversity was analyzed in samples collected from both vaccinated and nonvaccinated animals challenged with the H1N1 influenza A virus. Twenty-eight whole SIV genomes were obtained by next-generation sequencing, and differences in nucleotide variants between groups were established. Substitutions were allocated along all influenza genetic segments, while the most relevant nonsynonymous substitutions were allocated in the NS1 protein on samples collected from vaccinated animals, suggesting that SIV is continuously evolving despite vaccine application. Moreover, new viral variants were found in both vaccinated and nonvaccinated pigs, showing relevant substitutions in the HA, NA, and NP proteins, which may increase viral fitness under field conditions.

This study aimed to evaluate the immune response and protection correlates against influenza virus (IV) infection in pigs vaccinated with the novel NG34 HA1 vaccine candidate adjuvanted with either CAF^®01 or CDA/αGalCerMPEG (αGCM). Two groups of six pigs each were vaccinated intramuscularly twice with either NG34 + CAF^®01 or NG34 + CDA/αGCM. As controls, groups of animals (n = 6 or 4) either non-vaccinated or vaccinated with human seasonal trivalent influenza vaccine or NG34 + Freund’s adjuvant were included in the study. All animal groups were challenged with the 2009 pandemic (pdm09) strain of H1N1 (total amount of 7 × 10⁶ TCID₅₀/mL) via intranasal and endotracheal routes 21 days after second vaccination. Reduced consolidated lung lesions were observed both on days three and seven post-challenge in the animals vaccinated with NG34 + CAF^®01, whereas higher variability with relatively more severe lesions in pigs of the NG34 + CDA/αGCM group on day three post-infection. Among groups, animals vaccinated with NG34 + CDA/αGCM showed higher viral loads in the lung at seven days post infection whereas animals from NG34 + CAF^®01 completely abolished virus from the lower respiratory tract. Similarly, higher IFNγ secretion and stronger IgG responses against the NG34 peptide in sera was observed in animals from the NG34 + CAF^®01 group as compared to the NG34 + CDA/αGCM. NG34-vaccinated pigs with adjuvanted CAF^®01 or CDA/αGCM combinations resulted in different immune responses as well as outcomes in pathology and viral shedding.

Swine influenza viruses (SIVs), the causal agents of swine influenza, are not only important to control due to the economic losses in the swine industry, but also can be pandemic pathogens. Vaccination is one of the most relevant strategies to control and prevent influenza infection. Current human vaccines against influenza induce strain-specific immunity and annual update is required due to the virus antigenic shift phenomena. Previously, our group has reported the use of conserved hemagglutinin peptides (HA-peptides) derived from H1-influenza virus as a potential multivalent vaccine candidate. Immunization of swine with these HA-peptides elicited antibodies that recognized and neutralized heterologous influenza viruses in vitro and demonstrated strong hemagglutination-inhibiting activity. In the present work, we cloned one HA-peptide (named NG34) into a plasmid fused with cytotoxic T lymphocyte-associated antigen (CTLA4) which is a molecule that modifies T cell activation and with an adjuvant activity interfering with the adaptive immune response. The resulting plasmid, named pCMV-CTLA4-Ig-NG34, was administered twice to animals employing a needle-free delivery approach. Two studies were carried out to test the efficacy of pCMV-CTLA4-Ig-NG34 as a potential swine influenza vaccine, one in seronegative and another in seropositive pigs against SIV. The second one was aimed to evaluate whether pCMV-CTLA4-Ig-NG34 vaccination would overcome maternally derived antibodies (MDA). After immunization, all animals were intranasally challenged with an H3N2 influenza strain. A complete elimination or significant reduction in the viral shedding was observed within the first week after the challenge in the vaccinated animals from both studies. In addition, no challenged heterologous virus load was detected in the airways of vaccinated pigs. Overall, it is suggested that the pCMV-CTLA4-Ig-NG34 vaccine formulation could potentially be used as a multivalent vaccine against influenza viruses.

The immunization of poultry where H5 and H7 influenza viruses (IVs) are endemic is one of the strategies to prevent unexpected zoonoses. Our group has been focused on conserved HA-epitopes as potential vaccine candidates to obtain multivalent immune responses against distinct IV subtypes. In this study, two conserved epitopes (NG-34 and CS-17) fused to flagellin were produced in a Baculovirus platform based on Trichoplusia ni larvae as living biofactories. Soluble extracts obtained from larvae expressing "flagellin-NG34/CS17 antigen" were used to immunize chickens and the efficacy of the vaccine was evaluated against a heterologous H7N1 HPAIV challenge in chickens. The flagellin-NG34/CS17 vaccine protected the vaccinated chickens and blocked viral shedding orally and cloacally. Furthermore, no apparent clinical signs were monitored in 10/12 vaccinated individuals. The mechanism of protection conferred is under investigation.