Viral Immunology

Antimicrobial host defense peptides (AHDPs) are effective against a wide range of microbes, including viruses. The arteriviral infection caused by porcine reproductive and respiratory syndrome virus (PRRSV) is a devastating pandemic that causes the most economically significant disease of swine. We sought to determine if the expression of AHDPs was influenced by infection with PRRSV, and if porcine AHDPs have direct antiviral activity against PRRSV. Because pulmonary alveolar macrophages (PAMs) are primary targets of PRRSV infection, gene expression of porcine AHDPs was evaluated in lungs from fetal and 2-wk-old congenitally infected pigs. In PRRSV-positive lungs and PAMs, gene expression of most porcine AHDPs showed little upregulation. However, gene expression of porcine β-defensin-1 (pBD-1), pBD-4, pBD-104, pBD-123, and pBD-125 were downregulated more than threefold in 2-wk-old congenitally infected pig lungs. Incubation of PRRSV with pBD-3 or PG-4 significantly inhibited viral infectivity in MARC-145 cells. Using nine protegrin or protegrin-derived peptides, we determined that a cyclic analog of PG-4 increased anti-PRRSV activity, and that substitution of phenylalanine with valine eliminated most PG-4 antiviral activity. In PAMs, pBD-3 and PG-4 at 5–40 μg/mL consistently suppressed PRRSV titers. Collectively, these findings suggest a potential role for some porcine AHDPs as innate antiviral effectors in PRRSV infection. Moreover, modulation of porcine innate immune mechanisms with AHDPs may be one means of limiting the impact of this costly pandemic viral disease.

Vaccination with formalin-inactivated respiratory syncytial virus (RSV) vaccine results in enhanced respiratory tract inflammation and injury following subsequent RSV infection. RSV vaccine-enhanced disease can also be produced in mice by prior vaccination with a vaccinia virus vector containing the RSV G protein, followed by intranasal infectious RSV challenge, a process characterized by induction of a potent memory CD4⁺ T-cell response to challenge infection with some features characteristic of Th-2 CD4⁺ T-cell responses, including increased eosinophil accumulation in pulmonary inflammatory infiltrates. The adaptive immune response to the RSV G protein in immunized BALB/c mice is characterized by a weak or absent primary and secondary recall CD8⁺ T-cell response. These and related results have led to the hypothesis that the failure of the infected animals to mount an effective CD8⁺ memory T-cell (CD8⁺ Tm) response in this model could account for the pulmonary eosinophilia associated with the development of enhanced disease, and that CD8⁺ T cells may control the development of eosinophilia. In this study, we investigated how and when the generation of a CD8⁺ Tm response to RSV infection might affect the development of pulmonary eosinophilia in this model of vaccine-enhanced disease. By defining the CD8⁺ T-cell response kinetics and monitoring lung parenchymal eosinophil accumulation, we show that the establishment of an RSV-specific CD8⁺ Tm response in the infected lungs early after challenge infection (i.e., within the first 3 d of RSV infection) is necessary and sufficient to control pulmonary eosinophilia development. Additionally, our work suggests that the mechanism by which CD8⁺ T cells regulate this process is not by modulating the differentiation or development of the CD4⁺ Tm response. Rather, we demonstrate that IL-10 produced by early responding CD8⁺ Tm cells may regulate the pulmonary eosinophilia development observed in RSV vaccine-enhanced disease.

Hantaan virus (HTNV), the prototype member of the Hantavirus genus in the family Bunyaviridae, causes hemorrhagic fever with renal syndrome (HFRS), which is characterized by capillary leakage, hemorrhage, and renal injury, and is an important public health problem in China. Some kinds of immune cells, particularly CD8⁺ T cells, are involved in the pathogenesis of Hantavirus infection. The nucleocapsid protein (NP) of the Hantavirus is the most conserved structural protein and the most abundant viral protein produced during infection. It is one of the important target antigens that induce the CD8⁺ T-cell response. In this study, we examined the CD8⁺ T-cell response to HTNV NP C-terminal polypeptides. We synthesized 23 overlapping C-terminal polypeptides and detected the antigen-specific CD8⁺ T cell response in 15 patients with HFRS. The results demonstrated that there were NP-specific T-cell responses in bulk cultures of peripheral blood mononuclear cells (PBMCs) from 9 of 15 patients. The peptide 51 (aa 301–315: SPSSIWVFAGAPDRC), peptide 60 (aa 355–369: LRKKSSFYQSYLRRT), and peptide 70 (aa 415–429: DVKVKEISNQEPLKL) induced strong CD8⁺ T-cell responses. Among them, peptide 70 induced CTL responses in donors 7, 9, and 11, and the strongest responses were seen in donor 11. Depletion of CD8⁺ T cells from PBMCs completely abrogated the peptide-specific T-cell response, while depletion of CD4⁺ T cells did not diminish the number of IFN-γ spot-forming cells. These data suggest that infection with HTNV results in CTL responses to immunodominant regions on the NP.

The changes in peripheral blood mononuclear cells (PBMCs) have been studied in several reports in an attempt to determine the immune response against porcine reproductive and respiratory syndrome virus (PRRSV) infection. However, how these changes are evoked after PRRSV infection has not yet been clarified. The aim of this study was to analyze the changes seen in lymphocyte subsets and immunomodulatory cytokine expression in pigs after an acute experimental infection with a European PRRSV field isolate. Pigs were inoculated intramuscularly with PRRSV field isolate 2982. Samples from blood, medial retropharyngeal and tracheobronchial lymph nodes, and spleen were collected at different time points for flow cytometry studies and for cytokine expression by ELISA. CD21⁺ cell counts increased in PBMCs and tracheobronchial lymph node cells from 17 to 24 dpi, coinciding with an increase in PRRSV-specific antibody titer in blood. CD3⁺ T-cell counts increased mainly due to an enhancement of CD4⁻CD8^high and CD4⁺CD8⁺ T cells. CD4⁻CD8^low T cells were decreased in all organs studied, whereas CD4⁺CD8⁻ T cells decreased only in the spleen. The drop in viremia correlated with an enhancement of CD4⁻CD8^high T cells, and with a higher expression of interleukin-10 (IL-10) and interleukin-12 p40 (IL-12 p40). No efficient interferon-γ (IFN-γ) response was detected during the acute phase of the infection, and the expression of interferon-α (IFN-α) was late and reached its maximum expression once the viremia decreased. These results point to IL-10 and IL-12 as cytokines that might play a significant role in the PRRSV immune response, as may CD4⁻CD8^high T cells.

Highly pathogenic avian influenza H5N1 virus represents a growing threat for an influenza pandemic. Development of effective vaccines for H5N1 is a priority for pandemic preparedness. Focusing on influenza virus-like particles (VLPs) has been suggested as a promising vaccine approach. Recent VLP vaccination efforts have been concentrated on the H5N1 strains isolated from humans. Because all confirmed cases of human H5N1 infection were directly transmitted from infected poultry, it is of interest to develop VLP vaccines comprised of antigenic proteins of avian H5N1 strains in order to compare their efficacy in fighting diverse H5N1 strains with vaccines developed using human isolates. In this study, we generated a VLP vaccine composed of the HA, NA, and M1 proteins of the avian H5N1 influenza virus isolate A/chicken/Hubei/489/2004, which seems to occupy a unique phylogenetic position; it belongs to neither clade 1 nor clade 2. Upon infection of Sf9 insect cells using recombinant baculoviruses, the co-expressed HA, NA, and M1 proteins self-assembled and released into the culture medium as VLPs. In a mouse model, purified VLPs elicited an effective antibody response and conferred complete protection against heterologous human H5N1 influenza virus, as well as a homologous avian H5N1 influenza virus isolate. Our work provides further evidence that vaccination with influenza VLPs may be a productive approach to achieve protection against diverse H5N1 strains.