Recently emerged swine influenza A virus (H2N3) causes severe pneumonia in Cynomolgus macaques.

The triple reassortant H2N3 virus isolated from diseased pigs in the United States in 2006 is pathogenic for certain mammals without prior adaptation and transmits among swine and ferrets. Adaptation, in the H2 hemagglutinin derived from an avian virus, includes the ability to bind to the mammalian receptor, a significant prerequisite for infection of mammals, in particular humans, which poses a big concern for public health. Here we investigated the pathogenic potential of swine H2N3 in Cynomolgus macaques, a surrogate model for human influenza infection. In contrast to human H2N2 virus, which served as a control and largely caused mild pneumonia similar to seasonal influenza A viruses, the swine H2N3 virus was more pathogenic causing severe pneumonia in nonhuman primates. Both viruses replicated in the entire respiratory tract, but only swine H2N3 could be isolated from lung tissue on day 6 post infection. All animals cleared the infection whereas swine H2N3 infected macaques still presented with pathologic changes indicative of chronic pneumonia at day 14 post infection. Swine H2N3 virus was also detected to significantly higher titers in nasal and oral swabs indicating the potential for animal-to-animal transmission. Plasma levels of IL-6, IL-8, MCP-1 and IFNγ were significantly increased in swine H2N3 compared to human H2N2 infected animals supporting the previously published notion of increased IL-6 levels being a potential marker for severe influenza infections. In conclusion, the swine H2N3 virus represents a threat to humans with the potential for causing a larger outbreak in a non-immune or partially immune population. Furthermore, surveillance efforts in farmed pig populations need to become an integral part of any epidemic and pandemic influenza preparedness.

[Increase of immunogenicity of cold-adapted influenza vaccine by using adjuvant].

AIM: To assess increase of protective efficacy of live cold-adapted (ca) influenza vaccine after addition of adjuvant chitozan. MATERIALS AND METHODS: Used viruses: ca donor of attenuation A/Krasnodar/101/35/59 (H2N2) and epidemic strain A/Krasnodar/101/59 (H2N2); as an adjuvant--derivative of chitozan and microparticles of chitozan. Experiments were performed in outbred mice. Protective effect of immunization was measured by intranasal challenge by virulent strain of virus. Immune response was assessed by ELISA and indirect hemagglutination inhibition assay. RESULTS: During intranasal immunization of mice with intact CA donor of attenuation A/Krasnodar/101/35/59 (H2N2) addition of 1% solution of chitozan glutamate to vaccine material resulted in increased serum IgG in immunized mice and protective effect of immunization. Addition of adjuvant to ca donor strain did not influence on its ts-characteristic. It was shown that inactivated with ultraviolet radiation ca donor strain in combination with chitozan did not protect against infection caused by virulent strain A/Krasnodar/101/59, whereas the same doses of intact ca donor strain with chitozan were protective. Chitozan did not enhance replication of donor strain in upper respiratory tract of mice. CONCLUSION: Obtained data demonstrate that chitozan as a mucous-adhesive adjuvant could increase efficacy of live ca influenza vaccine.

Induction of broadly neutralizing H1N1 influenza antibodies by vaccination.

The rapid dissemination of the 2009 pandemic influenza virus underscores the need for universal influenza vaccines that elicit protective immunity to diverse viral strains. Here, we show that vaccination with plasmid DNA encoding H1N1 influenza hemagglutinin (HA) and boosting with seasonal vaccine or replication-defective adenovirus 5 vector encoding HA stimulated the production of broadly neutralizing influenza antibodies. This prime/boost combination increased the neutralization of diverse H1N1 strains dating from 1934 to 2007 as compared to either component alone and conferred protection against divergent H1N1 viruses in mice and ferrets. These antibodies were directed to the conserved stem region of HA and were also elicited in nonhuman primates. Cross-neutralization of H1N1 subtypes elicited by this approach provides a basis for the development of a universal influenza vaccine for humans.

Plasmacytoid dendritic cells enhance mortality during lethal influenza infections by eliminating virus-specific CD8 T cells.

Previous studies have shown that the reduction in CD8 T cell immunity observed during high-dose influenza A virus (IAV) infection is mediated via lymph node (LN) dendritic cells (DCs) that express Fas ligand (FasL) and drive FasL-Fas (DC-T)-induced apoptosis. However, the specific DC subset(s) within the LN and the additional factors required for DC-mediated elimination of IAV-specific CD8 T cells remain unknown. In this paper, we demonstrate that plasmacytoid DCs (pDCs), which downregulate FasL during sublethal, but not lethal, IAV infection, accumulate to greater numbers within the LNs of lethal dose-infected mice. Further our findings show that pDCs from lethal, but not sublethal, dose IAV infections drive elimination of Fas(+) CD8 T cells and that this elimination occurs only in the absence of TCR recognition of IAV peptide-MHC class I complexes. Together, these results suggest that pDCs play a heretofore unknown deleterious role during lethal dose IAV infections by limiting the CD8 T cell response.

Protective influenza-specific CD8 T cell responses require interactions with dendritic cells in the lungs.

Influenza infections induce a rapid, but transient, dendritic cell (DC) migration from the lungs to the lymph nodes (LNs) that is followed by substantial recruitment of DCs into the lungs without subsequent migration to the LNs. Given that peripheral DCs are primarily thought to be involved in the initiation of adaptive immunity after migration into lymphoid tissues, what role these newly lung-recruited DCs play in influenza virus immunity is unclear. In this study, we demonstrate that loss of non-LN migratory pulmonary DC subsets increases mortality, sustains higher viral titers, and impairs pulmonary CD8 T cell responses. Reconstitution of the lungs with pulmonary plasmacytoid DCs, CD8+ DCs, or interstitial DCs restores CD8 T cell responses in a cell contact-, major histocompatability complex I-, and influenza peptide-dependent manner. Thus, after their initial activation in the LN, protective influenza-specific CD8 T cell responses require additional antigen-dependent interactions, specifically with DCs in the lungs.

Effect of selection for phagocytosis in dwarf chickens on immune and reproductive characters.

In current study, phagocytosis product (PP) of peripheral blood monocytes was detected among 920 dwarf chickens (460 per sex) at 20 wk of age, and based on discrepancies of PP, the flock was grouped (the highest group, the medium group, and the lowest group). Then serum hemagglutination inhibition antibody titers and subpopulations of T-lymphocytes of each group were examined after inoculations of avian influenza virus H5N2 inactivated vaccine (20 wk of age), avian influenza virus H9 inactivated vaccine (24 wk of age), and Newcastle disease virus-egg drop syndrome virus bigeminal inactivated vaccine (28 wk of age), respectively, to study the relationship between PP and immune response. To gain insight into effects of selection for PP on number of eggs, mean egg weight, fertilization rate, hatchability, and rate of healthy chicks, 9 (3 x 3) mating combinations were conducted. The results showed that (1) selection for higher PP in both sexes benefited to humoral immunity but not CD8(+) T-lymphocyte mediated immunity in dwarf chickens; (2) there were effects of selection for higher PP in hens on fertilization rate (P < 0.05), hatchability (P < 0.05), rate of healthy chicks (P < 0.05), and level of IgY antibody (P < 0.0001); however, hens' PP had no effects on number of eggs (P > or = 0.05) or egg weight (P > or = 0.05) and cocks' PP had no effect (P > or = 0.05) on any trait mentioned above. The results indicated that phagocytosis of peripheral blood monocytes might be an indicator of humoral immunity in dwarf chickens; furthermore, selection of hens with higher PP was not only beneficial to fertilization rate, but also benefited to hatchability and rate of healthy chicks in that the hens had stronger humoral immunity, which might contribute to maternal antibody in eggs.

Early intrahepatic accumulation of CD8+ T cells provides a source of effectors for nonhepatic immune responses.

Interactions between the liver and CD8+ T cells can lead to tolerance, due in part to CD8+ T cell death. To test whether this was the case in an extrahepatic infection, we investigated the fate and effector capacity of intrahepatic CD8+ T cells during lung-restricted influenza infection in mice. Virus-specific T cells accumulated in livers without detectable intrahepatic presentation of viral Ags, and this accumulation was not restricted to the contraction phase, but was apparent as early as day 5. Intrahepatic influenza-specific cells were functionally similar to those recovered from the bronchioalveolar lavage, based on ex vivo cytokine production and specific target lysis. Both adoptive transfer of liver lymphocytes and orthotopic liver transplant of organs containing accumulated effector T cells revealed that activated CD8s from the liver were viable, expanded during reinfection, and generated a memory population that trafficked to lymphoid organs. Thus, intrahepatic CD8+ T cells re-enter circulation and generate functional memory, indicating that the liver does not uniformly incapacitate activated CD8+ T cells. Instead, it constitutes a substantial reservoir of usable Ag-specific effector CD8+ T cells involved in both acute and recall immune responses.

[Production of early cytokines after infection with A/Leningrad/134/57 (H2N2) wide-type virus and cold-adapted attenuated vaccine viruses].

The production of proinflammatory cytokines was studied following experimental infection of BALB/c mice with influenza viruses that differed in virulence. The generation of TNF-alpha, IL-6, IL-12, and IFN-gamma was investigated in the lung homogenates in the early periods after intranasal infection of mice with A/Leningrad/134/57 (H2N2) wild-type virus and cold-adapted attenuated vaccine viruses: A/Leningrad/134/17157 (H2N2) and A/Leningrad/134/47/57 (H2N2). Wild-type virus induced substantially higher levels of proinflammatory cytokines: TNF-alpha, IL-6, IL-12, and IFN-gamma. After infection with the cold-adapted viruses, the levels of the cytokines were reduced as compared to those induced by the wild-type virus. The A/Leningrad/134/47/57 virus was marked by a noticeable production of IL-6 and IFN-gamma in the murine lung, but it was less than with wild-type virus infection. At the same time, the more attenuated strain A/Leningrad/134/47/57 induced TNF-alpha and IFN-gamma in the quantities similar to those in the control animals. Thus, a response of proinflammatory cytokines in early infection in the murine lung depended on the level of viral replication in the lower respiratory tract and on the attenuation of influenza virus strains.

ELISpot assay as a sensitive tool to detect cellular immunity following influenza vaccination in kidney transplant recipients.

Enhanced cellular immunity following influenza vaccination has been undetectable in kidney transplant recipients so far. Protection from influenza is dependent on cellular and humoral immunity. Aim of the study was to investigate immune responses before and after vaccination with influenza A and B antigens in 65 kidney transplant recipients. A significant increase in proliferative responses was only observed towards influenza B (P < 0.0001) by lymphocyte transformation test. The enzyme-linked immunospot (ELISpot) assay was more sensitive and detected significant, 3- to 5-fold increases (P < 0.0001) in interferon-gamma secretion using influenza A and B antigens. Furthermore, influenza antibody titers increased significantly (P < 0.0001). At month 1 post-vaccination 85% of patients displayed specific cellular, and 95% or 92% humoral immunity against influenza A and B, respectively. Thus, applying the sensitive ELISpot assay, influenza-specific cellular immunity could be detected for the first time in kidney transplant recipients after vaccination.