No abstract

The emergence of multiple novel lineages of H1 and H3 influenza A viruses in swine has confounded control by inactivated vaccines. Because of substantial genetic and geographic heterogeneity among circulating swine influenza viruses, one vaccine strain per subtype cannot be efficacious against all of the current lineages. We have performed vaccination-challenge studies in pigs to examine whether priming and booster vaccinations with antigenically distinct H3N2 swine influenza viruses could broaden antibody responses and protection. We prepared monovalent whole inactivated, adjuvanted vaccines based on a European and a North American H3N2 swine influenza virus, which showed 81.5% aa homology in the HA1 region of the hemagglutinin and 83.4% in the neuraminidase. Our data show that (i) Priming with European and boosting with North American H3N2 swine influenza virus induces antibodies and protection against both vaccine strains, unlike prime-boost vaccination with a single virus or a single administration of bivalent vaccine. (ii) The heterologous prime-boost vaccination enhances hemagglutination inhibiting, virus neutralizing and neuraminidase inhibiting antibody responses against H3N2 viruses that are antigenically distinct from both vaccine strains. Antibody titers to the most divergent viruses were higher than after two administrations of bivalent vaccine. (iii) However, it does not induce antibodies to the conserved hemagglutinin stalk or to other hemagglutinin subtypes. We conclude that heterologous prime-boost vaccination might broaden protection to H3N2 swine influenza viruses and reduce the total amount of vaccine needed. This strategy holds potential for vaccination against influenza viruses from both humans and swine and for a better control of (reverse) zoonotic transmission of influenza viruses.

H9N2 avian influenza viruses are endemic in poultry in Asia and the Middle East. These viruses sporadically cause dead-end infections in pigs and humans raising concerns about their potential to adapt to mammals or reassort with human or swine influenza viruses. We performed ten serial passages with an avian H9N2 virus (A/quail/Hong Kong/G1/1997) in influenza naïve pigs to assess the potential of this virus to adapt to swine. Virus replication in the entire respiratory tract and nasal virus excretion were examined after each passage and we deep sequenced viral genomic RNA of the parental and passage four H9N2 virus isolated from the nasal mucosa and lung. The parental H9N2 virus caused a productive infection in pigs with a predominant tropism for the nasal mucosa, whereas only 50% lung samples were virus-positive. In contrast, inoculation of pigs with passage four virus resulted in viral replication in the entire respiratory tract. Subsequent passages were associated with reduced virus replication in the lungs and infectious virus was no longer detectable in the upper and lower respiratory tract of inoculated pigs at passage ten. The broader tissue tropism after four passages was associated with an amino acid residue substitution at position 225, within the receptor-binding site of the hemagglutinin. We also compared the parental H9N2, passage four H9N2 and the 2009 pandemic H1N1 (pH1N1) virus in a direct contact transmission experiment. Whereas only one out of six contact pigs showed nasal virus excretion of the wild-type H9N2 for more than four days, all six contact animals shed the passage four H9N2 virus. Nevertheless, the amount of excreted virus was significantly lower when compared to that of the pH1N1, which readily transmitted and replicated in all six contact animals. Our data demonstrate that serial passaging of H9N2 virus in pigs enhances its replication and transmissibility. However, full adaptation of an avian H9N2 virus to pigs likely requires an extensive set of mutations.

Avian H9N2 and 2009 pandemic H1N1 (pH1N1) influenza viruses can infect pigs and humans, raising the concern that H9N2:pH1N1 reassortant viruses could emerge. Such reassortants demonstrated increased replication and transmissibility in pig, but were still inefficient when compared to pH1N1. Here, we evaluated if a reassortant virus containing the hemagglutinin and neuraminidase of A/quail/Hong Kong/G1/1997 (H9N2) in the A/California/04/2009 (pH1N1) backbone could become better adapted to pigs by serial passaging. The tropism of the original H9N2:pH1N1 (P0) virus was restricted to the nasal mucosa, with no virus detected in the trachea or lungs. Nevertheless, after seven passages the H9N2:pH1N1 (P7) virus replicated in the entire respiratory tract. We also compared the transmissibility of H9N2:pH1N1 (P0), H9N2:pH1N1 (P7) and pH1N1. While only 2/6 direct-contact pigs showed nasal virus excretion of H9N2:pH1N1 (P0) ≥five days, 4/6 direct-contact animals shed the H9N2:pH1N1 (P7). Interestingly, those four animals shed virus with titers similar to those of the pH1N1, which readily transmitted to all six contact animals. The broader tissue tropism and the increased post-transmission replication after seven passages were associated with the HA-D225G substitution. Our data demonstrate that the pH1N1 internal-protein genes together with the serial passages favour H9N2 virus adaptation to pigs.

Effect of serial pig passages on the adaptation of an avian H9N2 influenza virus to swine. PLoS One 12(4), e0175267 (2017)' should read: 'Mancera Gracia, J. C., Van den Hoecke, S., Saelens, X. & Van Reeth, K. Effect of serial pig passages on the adaptation of an avian H9N2 influenza virus to swine. PLoS One 12(4), e0175267 (2017)'

Porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae (Mhyo) are important swine pathogens for which vaccination is a key control strategy. Three separate studies were performed to evaluate the duration of immunity (DOI) conferred by a novel vaccine combining PCV2a/PCV2b and Mhyo into a ready-to-use formulation. In each study, three-week-old naïve piglets were vaccinated (Day 0) and challenged 23-weeks later (Day 159) with either PCV2a, PCV2b or Mhyo. Pigs were euthanized three-to-four-weeks post-challenge. Vaccinated pigs had significantly lower PCV2 viremia from Day 168 until Day 175 (PCV2a study) or until euthanasia (PCV2b study), respectively. Fecal shedding was significantly lower for PCV2a-challenged from Day 171 until Day 178, and for PCV2b-challenged from Day 172 until euthanasia. In the PCV2a challenge study, there were no differences among vaccinates and controls in terms of percent of pigs positive for PCV2 immunohistochemistry, histiocytic replacement, or lymphoid depletion. However, significant differences for immunohistochemistry and histiocytic replacement, not lymphoid depletion, were observed among vaccinates and controls following PCV2b challenge. Vaccination supposed a significant reduction in the mean percentage of Mhyo-like lesions in the lung. Percentages of lung tissues positive for Mhyo via immunohistochemistry were 49.3% and 67.1% for vaccinated and control groups, respectively. One dose of the novel PCV2a/PCV2b/Mhyo vaccine conferred robust protection against challenge 23-weeks later for all three fractions.

This study aimed to evaluate the efficacy of a new trivalent vaccine containing inactivated Porcine Circovirus 1-2a and 1-2b chimeras and a Mycoplasma hyopneumoniae bacterin administered to pigs around 3 weeks of age. This trivalent vaccine has already been proved as efficacious in a split-dose regimen but has not been tested in a single-dose scenario. For this purpose, a total of four studies including two pre-clinical and two clinical studies were performed. Globally, a significant reduction in PCV-2 viraemia and faecal excretion was detected in vaccinated pigs compared to non-vaccinated animals, as well as lower histopathological lymphoid lesion plus PCV-2 immunohistochemistry scorings, and incidence of PCV-2-subclinical infection. Moreover, in field trial B, a significant increase in body weight and in average daily weight gain were detected in vaccinated animals compared to the non-vaccinated ones. Circulation of PCV-2b in field trial A and PCV-2a plus PCV-2d in field trial B was confirmed by virus sequencing. Hence, the efficacy of this new trivalent vaccine against a natural PCV-2a, PCV-2b or PCV-2d challenge was demonstrated in terms of reduction of histopathological lymphoid lesions and PCV-2 detection in tissues, serum and faeces, as well as improvement of production parameters.

Vaccines that protect against any H1N1 influenza A virus strain would be advantageous for use in pigs and humans. Here, we try to induce a pan-H1N1 antibody response in pigs by sequential vaccination with antigenically divergent H1N1 strains. Adjuvanted whole inactivated vaccines are given intramuscularly in various two- and three-dose regimens. Three doses of heterologous monovalent H1N1 vaccine result in seroprotective neutralizing antibodies against 71% of a diverse panel of human and swine H1 strains, detectable antibodies against 88% of strains, and sterile cross-clade immunity against two heterologous challenge strains. This strategy outperforms any two-dose regimen and is as good or better than giving three doses of matched trivalent vaccine. Neutralizing antibodies are H1-specific, and the second heterologous booster enhances reactivity with conserved epitopes in the HA head. We show that even the most traditional influenza vaccines can offer surprisingly broad protection, if they are administered in an alternative way.