Porcine Innate and Adaptative Immune Responses to Influenza and Coronavirus Infections

Antiviral Innate Immune Mechanisms

The innate immune response to viral infections includes cell‐mediated effector mechanisms such as natural killer (NK) activity, and soluble effectors among which type I interferons (IFN) play a major role. IFNs are a group of cytokines, initially identified by their ability to induce resistance to viral infection, ⁷ but also currently recognized as pro‐inflammatory molecules and potent modulators of both innate and adaptative immune responses. ⁸ , ⁹ In the course of an experimental influenza infection of pigs, type I IFN is present in the bronchoalveolar secretions together with tumor necrosis factor‐α (TNF‐α), and interleukins (IL‐1 and IL‐6). ¹⁰ The IFN response starts within 12 h post inoculation (PI) and lasts for several days. Peak cytokine titers occur within 18–24 h PI and they are correlated with the peak of virus replication, clinical signs, and infiltration of neutrophils in the bronchoalveolar lavage fluids. In addition, the level of lung pro‐inflammatory cytokines is correlated with the intensity of clinical signs. Thus, prior vaccination of pigs against influenza strongly reduced the production of infectious virus and cytokines in the lungs upon challenge, which was associated with clinical protection. ¹¹ IFN‐α producing leukocytes have been detected in the bronchiolar epithelium of infected pigs, at the peak of virus replication by immunohistochemistry. The IFN‐producing cells were found at very low numbers, and in close contact with influenza virus infected cells. ¹² An early type I IFN production was also characterized in coronavirus infected pigs, including at mucosal sites. Within 24 h after an experimental respiratory coronavirus infection (PRCV) in pigs, type I IFN started to be produced in the lung secretions, for more than 4 days, in the absence of significant levels of other pro‐inflammatory cytokines (TNF‐α and IL‐1). At the same time, there were few if any clinical signs and lung neutrophil infiltration was much less prominent than during a swine influenza infection, which suggests that lung type I IFN in itself is not involved in pro‐inflammatory and harmful effects. ¹³ The coronavirus TGEV experimental infection in newborn piglets is also characterized by a high and early IFN‐α production, in intestinal secretions, and in several other organs. ¹⁴ , ¹⁵ This coronavirus experimental infection model has generated original data pertaining to mechanisms of IFN‐α induction by viral glycoproteins: thus, only one viral external glycoprotein, gM, was shown to play a major IFN inducing role, ¹⁶ , ¹⁷ accordingly, virus‐like particles, made of only two TGEV proteins (M and E) and devoid of viral genome, were as effective as native virus to induce IFN‐α production by porcine leukocytes. ¹⁸ The IFN‐α producing cells, referred to as natural interferon producing cells (NIPC) ¹⁹ were recently identified as plasmacytoid dendritic cells (PDC), in both humans/mice ²⁰ , ²¹ and pigs. ²² PDC are low‐density cells, negative for CD11c and lineage markers (CD3, CD19, CD56, and CD14), but positive for MHCII, CD4, and CD123. ²³ In the course of coronavirus induced in vivo IFN‐α production in pigs, NIPC were detected in spleen and secondary lymphoid organs and shared several phenotypic features with PDC. ¹⁴ , ²⁴ Regarding mucosal innate responses, IFN‐producing cells were investigated in situ by immunohistochemical staining of duodenum, jejunum, ileum, mesenteric lymph node, popliteal lymph node, and spleen cryosections collected from TGEV‐infected piglets at the time of highest IFN production. This showed that the vast majority of IFN‐α producing cells were located in the small intestine (inside lamina propria and surrounding Peyer's patches) and accumulated in the mesenteric lymph nodes. ¹⁴ It was, therefore, concluded that most if not all circulating IFN‐α in TGEV‐infected piglets originates from gut and mesenteric lymph node. These intestinal IFN‐producing cells are in contact with but distinct from TGEV‐antigen positive cells and express MHCII, therefore resembling potential intestinal porcine pDC. Nevertheless, the frequency of intestinal NIPC is very low compared to ‘ordinary’ DC that are extremely numerous, sometimes filling the whole lamina propria of a villus. ²⁵ One can wonder about their function at such site: their small number makes them unlikely to be a major antigen‐presenting DC subset. On the other hand, even a rare intestinal NIPC in mesenteric lymph node will flood the T cell area with IFN‐α which is very likely to influence the outcome of the immune response. Contrary to their murine counterpart but in common with humans, porcine NIPC/pDC are the only DC subset able to bind bacterial/viral components via TLR9, TLR7 or yet unknown receptors. ²⁶ Mucosal porcine NIPC/pDC could therefore be preferential targets for using natural ligands of TLR9 (bacterial and viral DNA or CpG‐ODN ²⁷ ) as immunomodulators and IFN inducers. Given the potent IFN mediated antiviral and immunomodulatory effects, ⁷ the use of IFN or IFN inducers may prove an efficient strategy ²⁸ for a better control of influenza virus and coronavirus infections in pigs. Besides type I IFN, other antiviral innate immune mechanisms include NK cell activity and both influenza virus and coronavirus were shown to activate porcine NK activity. ²⁹

Antiviral Adaptative Immune Mechanisms at Respiratory Surfaces

Antiviral adaptative immune mechanisms involve neutralizing antibody (Ab), including secretory IgA at mucosal surfaces, and cytotoxic T lymphocytes (CTL). Because influenza virus and coronaviruses target mucosal surfaces, adaptative immune responses have to be specifically characterized at mucosal sites. Thus, nasal and pulmonary antibody responses were analyzed in influenza virus infected or vaccinated pigs, showing short‐lived IgG, but more durable IgA local Ab responses, ³⁰ , ³¹ which resulted in protection to virus challenge. ³² Interestingly, primary influenza virus infection in pigs induced local antigen‐specific lymphoproliferative responses ³³ and a long‐lived increase of lung CD8⁺ T cells which could play a role in the broad‐spectrum immune protection to heterotypic virus strains. ³⁴ Pigs infected by a PRCV showed virus‐specific IgG Ab‐secreting cells and lymphoproliferative responses in the bronchial lymph nodes with low Ab responses in the gut, whereas the TGEV induced more IgA Ab‐secreting cells in gut tissues and T cell responses in mesenteric lymph nodes, which illustrates the importance of the route of antigen or vaccine administration for inducing local immune effector mechanisms. ³⁵ , ³⁶

These results show that specific antiviral effector mechanisms, including IgG and IgA Ab production, lymphoproliferative responses and CD8⁺ T cell (presumably CTL) recruitment, are induced at mucosal sites of virus replication following infection and/or vaccination, with either influenza virus or coronavirus, in the pig respiratory tract. Local immunity is more appropriate for controlling virus spreading and airborne transmission from animals to animals and from animal reservoirs to human targets.