Alterations of the immune response to influenza virus by immunotherapy and infection

Respiratory tract infections and parasite infections are common and have a significant impact on global health and economy. Influenza virus is one of the most common causes of upper respiratory tract infection, infecting up to one billion people each year. Due to constant mutations in antigenic surface proteins, long- lasting protective immunity against subsequent infections is hard to achieve by natural infection and/or vaccination. Vaccines against seasonal influenza viruses are available but waning protection remains a challenge. Thus, other strategies to increase protection against influenza virus disease are needed. In addition, mammalian and avian influenza viruses can exchange genetic material within a host leading to generation of a new virus with potential to cause a pandemic. Twenty percent of the global population are infected with soil-transmitted helminths. The highest prevalences overlaps with the geographical areas where mortality due to respiratory tract infections, including influenza virus, is the highest. Experimentally it has been demonstrated that helminth infections can reduce the host immune response to vaccination and may ameliorate or aggravate disease induced by another pathogen. To date, little is known about the effects of a helminth infection on pre-existing immunity.

The aim of this PhD project was to investigate how the development and maintenance of influenza virus immunity was affected by prophylactic immunotherapy with avian antibodies against influenza virus or intestinal helminth infection with Heligmosomoides polygyrus.

To this end, mouse models were used to study the adaptive immune response against influenza virus. In Paper I, mice were infected with influenza virus and at different timepoints the mice were euthanised for analysis of the adaptive immune response in mediastinal lymph nodes (MLN), lungs, spleens, inguinal lymph nodes (ILN), and mesenteric lymph nodes (MesLN). In Paper II, mice were treated intranasally with influenza virus-specific IgY (IgY anti-H5N1) prior to and after influenza virus infection. Influenza virus-specific T cells were quantified in MLN, lungs, and spleens. To investigate the quality of the immunological memory, the mice received a second infection with homologous or heterologous influenza virus. In Paper III, the mice were infected with the intestinal nematode Heligmosomoides polygyrus (H. polygyrus) three weeks after influenza virus infection. At the end of the experiment, influenza virus-specific T cells were quantified by flow cytometry. In addition, gene expression and TCR repertoire analysis were performed on influenza virus-specific CD8+ T cells after trickle infection with H. polygyrus.

In Paper I, the kinetics of the T cell response to influenza virus in mice, including CD4+ Trm and three subsets of CD8+ Trm were determined. The adaptive immune response was analysed in lungs and respiratory tract-draining MLN, as well as in spleen, ILN, and MesLN. Using CD69 as a marker of activation we observed a biphasic activation curve with local peaks at days three and 8 - 12 post infection among CD4+ T cells in all organs studied. CD8+ T cells in lungs and ILN displayed the same pattern whereas only a peak at three days post infection was observed in MLN, spleen, and MesLN. We hypothesized that the second peak observed in lungs was due to expansion of lung-resident T cells (Trm). During active infection and early after viral clearance, CD103-CD49a+ CD8+ Trm was the dominant Trm subset whereas CD103-CD49+ CD8+ Trm was the most abundant subset during the memory phase. To summarise, even though influenza virus is restricted to the respiratory tract, the infection elicits a systemic T cell response, albeit more pronounced in respiratory tract-associated tissues with local expansion of different Trm subsets at distinct stages after infection.

In Paper II the impact on development of host immunity induced by influenza virus infection after passive immunization with influenza virus-specific avian IgY was investigated. The results demonstrated that protective homologous and heterologous immunity developed in influenza virus-infected mice receiving prophylactic IgY anti-H5N1. First, it was confirmed that prophylactic administration of influenza virus-specific IgY resulted in protection against disease symptoms and severe pneumonia after infection with influenza virus. Despite having a lower magnitude of the influenza virus-specific T cell response during acute infection, there was no significant difference after clearance of infection. Upon reinfection at 35 days post infection, mice previously treated with IgY anti-H5N1 were equally protected as mice not treated with IgY anti-H5N1 during primary infection. Nevertheless, challenge infection with heterologous influenza virus at 35 days post infection or homologous virus at three months post infection resulted in a minor and transient weight loss in the mice treated with IgY anti-H5N1. In addition, we demonstrated that repeated administration of IgY anti- H5N1 could prolong the survival in immunodeficient CB-17 SCID mice after influenza virus infection, indicating that passive immunization can be used to protect individuals with a severely impaired immune system. Taken together, prophylactic antibody therapy protects against disease while allowing for development of protective immunity against severe disease and death after re- exposure to homologous or heterologous influenza virus.

The effects of infection with H. polygyrus on pre-existing cellular immunity against influenza virus was investigated in Paper III. It was found that mice infected with influenza virus and then with H. polygyrus ("co-infected") had a reduction in influenza virus-specific T cells at seven weeks post infection. Nevertheless, after exposure to a second infection with homologous influenza virus two months post primary infection, co-infected mice had the same level of protective immunity as mice previously infected with influenza virus only. In contrast, when challenged at four months post primary infection, co-infected mice presented with a transient, minor weight loss indicating a reduction in potency of immunity.

To potentiate the effects worms and drive a strong immune response against another infection, repeated H. polygyrus infections were given. After trickle infection with H. polygyrus, genes associated with protein folding and immune function were differentially expressed in total and virus-specific CD8+ T cells of co-infected mice and mice infected with influenza virus only. Nevertheless, infection with H. polygyrus did not significantly affect the TCR repertoire of influenza virus-specific CD8+ T cells. The findings indicate that H. polygyrus causes a reduction and transcriptional changes in antigen-specific CD8+ T cells, which in turn may accelerate waning of the immunological memory against influenza virus.

To summarize, influenza virus elicits a robust local immune response but also activates B cells and T cells in secondary lymphoid organs distant the site of infection. After resolution of infection, humoral and cellular immunity including CD103+CD49a+ CD8+ Trm is developed that confers protection against homologous and heterologous influenza viruses. Both prophylactic antibody therapy and intestinal helminth infections can modulate cellular immunity against influenza virus which manifests as waning immunity, which still protected the mice against severe disease. Nevertheless, treatment with IgY anti-H5N1 protected against influenza virus disease, reduced pulmonary inflammation, and allowed for generation of protective immunity. This makes prophylactic antibody therapy a suitable option and/or complement to influenza virus vaccination. In contrast, infection with H. polygyrus reduces the numbers of pre-existing antigen-specific T cells and can induce transcriptional alterations in influenza virus-specific CD8+ T cells. This can be taken into consideration when planning vaccination schedules in areas with high burden of helminth infections.

List of scientific papers

I. T cell kinetics reveal expansion of distinct lung T cell subsets in acute versus in resolved influenza virus infection. Eriksson, M., Nylén, S., Grönvik, K-O. Frontiers in Immunology. (2022) 13: 949299. https://doi.org/10.3389/fimmu.2022.949299

II. Passive immunization of mice with IgY anti-H5N1 protects against experimental influenza virus infection and allows development of protective immunity. Eriksson, M., Nylén, S., Grönvik, K-O. Vaccine. (2024), 42, 25: 126133. https://doi.org/10.1016/j.vaccine.2024.07.034

III. Impact of Heligmosomoides polygyrus on influenza virus- specific T cell immunity in mice Eriksson, M., Grönvik, K-O., Åbrink, M., Nylén, S. [Manuscript]