Alveolar Macrophages Are Essential for Protection from Respiratory Failure and Associated Morbidity following Influenza Virus Infection

Alveolar macrophages (AM) are critical for defense against bacterial and fungal infections. However, a definitive role of AM in viral infections remains unclear. We here report that AM play a key role in survival to influenza and vaccinia virus infection by maintaining lung function and thereby protecting from asphyxiation. Absence of AM in GM-CSF-deficient (Csf2 ^−/−) mice or selective AM depletion in wild-type mice resulted in impaired gas exchange and fatal hypoxia associated with severe morbidity to influenza virus infection, while viral clearance was affected moderately. Virus-induced morbidity was far more severe in Csf2 ^−/− mice lacking AM, as compared to Batf3-deficient mice lacking CD8α⁺ and CD103⁺ DCs. Csf2 ^−/− mice showed intact anti-viral CD8⁺ T cell responses despite slightly impaired CD103⁺ DC development. Importantly, selective reconstitution of AM development in Csf2rb ^−/− mice by neonatal transfer of wild-type AM progenitors prevented severe morbidity and mortality, demonstrating that absence of AM alone is responsible for disease severity in mice lacking GM-CSF or its receptor. In addition, CD11c-Cre/Pparg ^fl/fl mice with a defect in AM but normal adaptive immunity showed increased morbidity and lung failure to influenza virus. Taken together, our results suggest a superior role of AM compared to CD103⁺ DCs in protection from acute influenza and vaccinia virus infection-induced morbidity and mortality.     

The expression of FOXP3 in lesions of several forms of leprosy in patients co-infected with HIV

BackgroundBrazil remains endemic for infection by the human immunodeficiency virus (HIV) and leprosy, having a major impact on public health and the life quality of affected patients. Although the relevance of this co-infection is recognized, several aspects, such as the immune response, are not yet fully understood. The objective of this study was to investigate the expression of FOXP3⁺ Treg cells in leprosy skin lesions and to correlate their clinical forms, laboratory characteristics (CD4, CD8, and CV), and the immune reconstitution syndrome in HIV-leprosy co-infection.Methodology/Principal findingsAn observational, cross-sectional, and analytical study was carried out comparing four groups of patients: those with concomitant diagnosis of leprosy and HIV infection without a leprosy reaction, those with leprosy and HIV co-infection patients with a reverse reaction (RR), those with leprosy without HIV and without reaction, and those with leprosywithout HIV and with RR. The patients were diagnosed at a dermatology outpatient clinic located in Belém, Pará, Brazil, from 2003 to 2017. In the sample studied, there was a positive correlation between FOXP3⁺ cell density and viral load, negative correlation with blood CD4⁺ (not statistically significant), significant positive correlation in CD8 count in patients with leprosy reaction, and positive relationship in patients with IRIS. The density of cells expressing FOXP3 was higher in the BL/LL forms in patients without HIV, although the difference was not statistically significant. However, the cell mean was higher in the TT/BT forms in patients co-infected with leprosy and HIV, showing contradictory results.Conclusions/SignificanceThese findings support that higher activity of the HIV may stimulate or result in a higher expression of FOXP3-Tregs and that they may be involved in active immunosuppression observed at the infection site at the tissue level. This supports the need to expand studies on FOXP3⁺ Treg cells in co-infected patients.     

HIV Replication Is Not Controlled by CD8+ T Cells during the Acute Phase of the Infection in Humanized Mice

HIV replication follows a well-defined pattern during the acute phase of the infection in humans. After reaching a peak during the first few weeks after infection, viral replication resolves to a set-point thereafter. There are still uncertainties regarding the contribution of CD8⁺ T cells in establishing this set-point. An alternative explanation, supported by in silico modeling, would imply that viral replication is limited by the number of available targets for infection, i.e. CD4⁺CCR5⁺ T cells. Here, we used NOD.SCID.gc^-/- mice bearing human CD4⁺CCR5⁺ and CD8⁺ T cells derived from CD34⁺ progenitors to investigate the relative contribution of both in viral control after the peak. Using low dose of a CCR5-tropic HIV virus, we observed an increase in viral replication followed by “spontaneous” resolution of the peak, similar to humans. To rule out any possible role for CD8⁺ T cells in viral control, we infected mice in which CD8⁺ T cells had been removed by a depleting antibody. Globally, viral replication was not affected by the absence of CD8⁺ T cells. Strikingly, resolution of the viral peak was equally observed in mice with or without CD8⁺ T cells, showing that CD8⁺ T cells were not involved in viral control in the early phase of the infection. In contrast, a marked and specific loss of CCR5-expressing CD4⁺ T cells was observed in the spleen and in the bone marrow, but not in the blood, of infected animals. Our results strongly suggest that viral replication during the acute phase of the infection in humanized mice is mainly constrained by the number of available targets in lymphoid tissues rather than by CD8⁺ T cells.     

Ascaris co-infection does not alter malaria-induced anaemia in a cohort of Nigerian preschool children

Background

Co-infection with malaria and intestinal parasites such as Ascaris lumbricoides is common. Malaria parasites induce a pro-inflammatory immune response that contributes to the pathogenic sequelae, such as malarial anaemia, that occur in malaria infection. Ascaris is known to create an anti-inflammatory immune environment which could, in theory, counteract the anti-malarial inflammatory immune response, minimizing the severity of malarial anaemia. This study examined whether Ascaris co-infection can minimize the severity of malarial anaemia.

Methods

Data from a randomized controlled trial on the effect of antihelminthic treatment in Nigerian preschool-aged (6–59 months) children conducted in 2006–2007 were analysed to examine the effect of malaria and Ascaris co-infection on anaemia severity. Children were enrolled and tested for malaria, helminths and anaemia at baseline, four, and eight months. Six hundred and ninety subjects were analysed in this study. Generalized linear mixed models were used to assess the relationship between infection status and Ascaris and Plasmodium parasite intensity on severity of anaemia, defined as a haemoglobin less than 11 g/dL.

Results

Malaria prevalence ranged from 35-78% over the course of this study. Of the malaria-infected children, 55% were co-infected with Ascaris at baseline, 60% were co-infected four months later and 48% were co-infected eight months later, underlining the persistent prevalence of malaria-nematode co-infections in this population. Over the course of the study the percentage of anaemic subjects in the population ranged between 84% at baseline and 77% at the eight-month time point. The odds of being anaemic were four to five times higher in children infected with malaria compared to those without malaria. Ascaris infection alone did not increase the odds of being anaemic, indicating that malaria was the main cause of anaemia in this population. There was no significant difference in the severity of anaemia between children singly infected with malaria and co-infected with malaria and Ascaris.

Conclusion

In this cohort of Nigerian preschool children, malaria infection was the major contributor to anaemia status. Ascaris co-infection neither exacerbated nor ameliorated the severity of malarial anaemia.     

Clinical profile and mortality in patients with T. cruzi/HIV co-infection from the multicenter data base of the “Network for healthcare and study of Trypanosoma cruzi/HIV co-infection and other immunosuppression conditions”

ObjectiveChagas disease (CD) globalization facilitated the co-infection with Human Immunodeficiency Virus (HIV) in endemic and non-endemic areas. Considering the underestimation of Trypanosoma cruzi (T. cruzi)-HIV co-infection and the risk of life-threatening Chagas Disease Reactivation (CDR), this study aimed to analyze the major co-infection clinical characteristics and its mortality rates.MethodsThis is a cross-sectional retrospective multicenter study of patients with CD confirmed by two serological or one parasitological tests, and HIV infection confirmed by immunoblot. CDR was diagnosed by direct microscopy with detection of trypomastigote forms in the blood or other biological fluids and/or amastigote forms in inflammatory lesions.ResultsOut of 241 patients with co-infection, 86.7% were from Brazil, 47.5% had <200 CD4⁺ T cells/μL and median viral load was 17,000 copies/μL. Sixty CDR cases were observed. Death was more frequent in patients with reactivation and was mainly caused by CDR. Other causes of death unrelated to CDR were the manifestation of opportunistic infections in those with Acquired Immunodeficiency Syndrome. The time between the co-infection diagnosis to death was shorter in patients with CDR. Lower CD4⁺ cells count at co-infection diagnosis was independently associated with reactivation. Similarly, lower CD4⁺ cells numbers at co-infection diagnosis and male sex were associated with higher lethality in CDR. Additionally, CD4⁺ cells were lower in meningoencephalitis than in myocarditis and milder forms.ConclusionThis study showed major features on T. cruzi-HIV co-infection and highlighted the prognostic role of CD4⁺ cells for reactivation and mortality. Since lethality was high in meningoencephalitis and all untreated patients died shortly after the diagnosis, early diagnosis, immediate antiparasitic treatment, patient follow-up and epidemiological surveillance are essentials in T. cruzi/HIV co-infection and CDR managements.     

Attenuated and Replication-Competent Vaccinia Virus Strains M65 and M101 with Distinct Biology and Immunogenicity as Potential Vaccine Candidates against Pathogens

Replication-competent poxvirus vectors with an attenuation phenotype and with a high immunogenic capacity of the foreign expressed antigen are being pursued as novel vaccine vectors against different pathogens. In this investigation, we have examined the replication and immunogenic characteristics of two vaccinia virus (VACV) mutants, M65 and M101. These mutants were generated after 65 and 101 serial passages of persistently infected Friend erythroleukemia (FEL) cells. In cultured cells of different origins, the mutants are replication competent and have growth kinetics similar to or slightly reduced in comparison with those of the parental Western Reserve (WR) virus strain. In normal and immune-suppressed infected mice, the mutants showed different levels of attenuation and pathogenicity in comparison with WR and modified vaccinia Ankara (MVA) strains. Wide genome analysis after deep sequencing revealed selected genomic deletions and mutations in a number of viral open reading frames (ORFs). Mice immunized in a DNA prime/mutant boost regimen with viral vectors expressing the LACK (Leishmania homologue for receptors of activated C kinase) antigen of Leishmania infantum showed protection or a delay in the onset of cutaneous leishmaniasis. Protection was similar to that triggered by MVA-LACK. In immunized mice, both polyfunctional CD4⁺ and CD8⁺ T cells with an effector memory phenotype were activated by the two mutants, but the DNA-LACK/M65-LACK protocol preferentially induced CD4⁺ whereas DNA-LACK/M101-LACK preferentially induced CD8⁺ T cell responses. Altogether, our findings showed the adaptive changes of the WR genome during long-term virus-host cell interaction and how the replication competency of M65 and M101 mutants confers distinct biological properties and immunogenicity in mice compared to those of the MVA strain. These mutants could have applicability for understanding VACV biology and as potential vaccine vectors against pathogens and tumors.     

Attrition of TCR Vα7.2+ CD161++ MAIT Cells in HIV-Tuberculosis Co-Infection Is Associated with Elevated Levels of PD-1 Expression

Mucosal-associated invariant T (MAIT) cells are evolutionarily conserved antimicrobial MR1-restricted CD8⁺ T cells co-expressing the semi-invariant TCR Vα7.2, and are numerous in the blood and mucosal tissues of humans. MAIT cells appear to undergo exhaustion in chronic viral infections. However, their role in human immunodeficiency virus type 1 (HIV-1) mono-infection and HIV/tuberculosis (TB) co-infection have seldom been elaborately investigated. We conducted a cross-sectional study to investigate the frequencies and phenotypes of CD161⁺⁺CD8⁺ T cells among anti-retroviral therapy (ART)/anti-TB therapy (ATT) treatment-naïve HIV/TB co-infected, ART/TB treated HIV/TB co-infected, ART naïve HIV-infected, ART-treated HIV-infected patients, and HIV negative healthy controls (HCs) by flow cytometry. Our data revealed that the frequency of MAIT cells was severely depleted in HIV mono- and HIV/TB co-infections. Further, PD-1 expression on MAIT cells was significantly increased in HIV mono- and HIV-TB co-infected patients. The frequency of MAIT cells did not show any significant increase despite the initiation of ART and/or ATT. Majority of the MAIT cells in HCs showed a significant increase in CCR6 expression as compared to HIV/TB co-infections. No marked difference was seen with expressions of chemokine co-receptor CCR5 and CD103 among the study groups. Decrease of CCR6 expression appears to explain why HIV-infected patients display weakened mucosal immune responses.     

Allergic Airway Disease in Mice Alters T and B Cell Responses during an Acute Respiratory Poxvirus Infection

Pulmonary viral infections can exacerbate or trigger the development of allergic airway diseases via multiple mechanisms depending upon the infectious agent. Respiratory vaccinia virus transmission is well established, yet the effects of allergic airway disease on the host response to intra-pulmonary vaccinia virus infection remain poorly defined. As shown here BALB/c mice with preexisting airway disease infected with vaccinia virus developed more severe pulmonary inflammation, higher lung virus titers and greater weight loss compared with mice inoculated with virus alone. This enhanced viremia was observed despite increased pulmonary recruitment of CD8⁺ T effectors, greater IFNγ production in the lung, and high serum levels of anti-viral antibodies. Notably, flow cytometric analyses of lung CD8⁺ T cells revealed a shift in the hierarchy of immunodominant viral epitopes in virus inoculated mice with allergic airway disease compared to mice treated with virus only. Pulmonary IL-10 production by T cells and antigen presenting cells was detected following virus inoculation of animals and increased dramatically in allergic mice exposed to virus. IL-10 modulation of host responses to this respiratory virus infection was greatly influenced by the localized pulmonary microenvironment. Thus, blocking IL-10 signaling in virus-infected mice with allergic airway disease enhanced pulmonary CD4⁺ T cell production of IFNγ and increased serum anti-viral IgG1 levels. In contrast, pulmonary IFNγ and virus-specific IgG1 levels were reduced in vaccinia virus-treated mice with IL-10 receptor blockade. These observations demonstrate that pre-existing allergic lung disease alters the quality and magnitude of immune responses to respiratory poxviruses through an IL-10-dependent mechanism.     

The beta2 integrin CD11c distinguishes a subset of cytotoxic pulmonary T cells with potent antiviral effects in vitro and in vivo

Background

The integrin CD11c is known as a marker for dendritic cells and has recently been described on T cells following lymphotropic choriomeningitis virus infection, a systemic infection affecting a multitude of organs. Here, we characterise CD11c bearing T cells in a murine model of localised pulmonary infection with respiratory syncytial virus (RSV).

Methods

Mice were infected intranasally with RSV and expression of β2 integrins and T lymphocyte activation markers were monitored by flow cytometry. On day 8 post RSV infection CD11c⁺ CD8⁺ and CD11c^- CD8⁺ T cells were assessed for cytokine production, cytotoxic activity and migration. Expression of CD11c mRNA in CD8⁺ T cells was assessed by quantitative PCR.

Results

Following RSV infection CD11c⁺ CD8⁺ T cells were detectable in the lung from day 4 onwards and accounted for 45.9 ± 4.8% of CD8⁺ T cells on day 8 post infection, while only few such cells were present in mediastinal lymph nodes, spleen and blood. While CD11c was virtually absent from CD8⁺ T cells in the absence of RSV infection, its mRNA was expressed in CD8⁺ T cells of both naïve and RSV infected mice. CD11c⁺, but not CD11c^-, CD8⁺ T cells showed signs of recent activation, including up-regulation of CD11a and expression of CD11b and CD69 and were recruited preferentially to the lung. In addition, CD11c⁺ CD8⁺ T cells were the major subset responsible for IFNγ production, induction of target cell apoptosis in vitro and reduction of viral titres in vivo.

Conclusion

CD11c is a useful marker for detection and isolation of pulmonary antiviral cytotoxic T cells following RSV infection. It identifies a subset of activated, virus-specific, cytotoxic T cells that exhibit potent antiviral effects in vivo.     

Smallpox vaccination induces a substantial increase in commensal skin bacteria that promote pathology and influence the host response

Interactions between pathogens, host microbiota and the immune system influence many physiological and pathological processes. In the 20^th century, widespread dermal vaccination with vaccinia virus (VACV) led to the eradication of smallpox but how VACV interacts with the microbiota and whether this influences the efficacy of vaccination are largely unknown. Here we report that intradermal vaccination with VACV induces a large increase in the number of commensal bacteria in infected tissue, which enhance recruitment of inflammatory cells, promote tissue damage and influence the host response. Treatment of vaccinated specific-pathogen-free (SPF) mice with antibiotic, or infection of genetically-matched germ-free (GF) animals caused smaller lesions without alteration in virus titre. Tissue damage correlated with enhanced neutrophil and T cell infiltration and levels of pro-inflammatory tissue cytokines and chemokines. One month after vaccination, GF and both groups of SPF mice had equal numbers of VACV-specific CD8⁺ T cells and were protected from disease induced by VACV challenge, despite lower levels of VACV-neutralising antibodies observed in GF animals. Thus, skin microbiota may provide an adjuvant-like stimulus during vaccination with VACV and influence the host response to vaccination.     

Investigation of Pathogenesis of H1N1 Influenza Virus and Swine Streptococcus suis Serotype 2 Co-Infection in Pigs by Microarray Analysis

Swine influenza virus and Streptococcus suis are two important contributors to the porcine respiratory disease complex, and both have significant economic impacts. Clinically, influenza virus and Streptococcus suis co-infections in pigs are very common, which often contribute to severe pneumonia and can increase the mortality. However, the co-infection pathogenesis in pigs is unclear. In the present study, co-infection experiments were performed using swine H1N1 influenza virus and Streptococcus suis serotype 2 (SS2). The H1N1-SS2 co-infected pigs exhibited more severe clinical symptoms, serious pathological changes, and robust apoptosis of lungs at 6 days post-infection compared with separate H1N1 and SS2 infections. A comprehensive gene expression profiling using a microarray approach was performed to investigate the global host responses of swine lungs against the swine H1N1 infection, SS2 infection, co-infection, and phosphate-buffered saline control. Results showed 457, 411, and 844 differentially expressed genes in the H1N1, SS2, and H1N1-SS2 groups, respectively, compared with the control. Noticeably, genes associated with the immune, inflammatory, and apoptosis responses were highly overexpressed in the co-infected group. Pathway analysis indicated that the cytokine–cytokine receptor interactions, MAPK, toll-like receptor, complement and coagulation cascades, antigen processing and presentation, and apoptosis pathway were significantly regulated in the co-infected group. However, the genes related to these were less regulated in the separate H1N1 and SS2 infection groups. This observation suggested that a certain level of synergy was induced by H1N1 and SS2 co-infection with significantly stronger inflammatory and apoptosis responses, which may lead to more serious respiratory disease syndrome and pulmonary pathological lesion.     

CD3+/TCR-αβ− Cells Are Important in Protecting Spinal Cord Tissues against Theiler's Virus Strain GD VII Infection

Intravenous infection with Theiler's virus strain GD VII causes acute encephalomyelitis in mice. Endogenous IFN-γ produced in the spinal cord is important to protect the tissue in mice infected with this virus. Neither CD4⁺ cells nor CD8⁺ cells infiltrated the spinal cords of infected mice until Day 9 postinfection. However, the number of CD3⁺/TCR-γδ⁺ cells increased in the spinal cords of mice infected with the virus. These cells resided in the spinal cords of normal mice, and produced IFN-γ as a result of stimulation by immobilized anti-CD3 mAb. Elimination of CD3⁺ cells by the administration of a specific mAb augmented viral replication and suppressed production of endogenous IFN-γ. Depletion of TCR-αβ⁺ cells and ASGM1⁺ cells did not affect the viral replication, and did not alter the production of IFN-γ. Therefore, CD3⁺/TCR-αβ^– cells producing IFN-γ play an important role in the protection of the spinal cord against Theiler's virus infection. These results suggest that CD3⁺/TCR-αβ^– cells might be identical to TCR-γδ⁺ cells.     

Immunodomination during Peripheral Vaccinia Virus Infection

Immunodominance is a fundamental property of CD8⁺ T cell responses to viruses and vaccines. It had been observed that route of administration alters immunodominance after vaccinia virus (VACV) infection, but only a few epitopes were examined and no mechanism was provided. We re-visited this issue, examining a panel of 15 VACV epitopes and four routes, namely intradermal (i.d.), subcutaneous (s.c.), intraperitoneal (i.p.) and intravenous (i.v.) injection. We found that immunodominance is sharpened following peripheral routes of infection (i.d. and s.c.) compared with those that allow systemic virus dissemination (i.p. and i.v.). This increased immunodominance was demonstrated with native epitopes of VACV and with herpes simplex virus glycoprotein B when expressed from VACV. Responses to some subdominant epitopes were altered by as much as fourfold. Tracking of virus, examination of priming sites, and experiments restricting virus spread showed that priming of CD8⁺ T cells in the spleen was necessary, but not sufficient to broaden responses. Further, we directly demonstrated that immunodomination occurs more readily when priming is mainly in lymph nodes. Finally, we were able to reduce immunodominance after i.d., but not i.p. infection, using a VACV expressing the costimulators CD80 (B7-1) and CD86 (B7-2), which is notable because VACV-based vaccines incorporating these molecules are in clinical trials. Taken together, our data indicate that resources for CD8⁺ T cell priming are limiting in local draining lymph nodes, leading to greater immunodomination. Further, we provide evidence that costimulation can be a limiting factor that contributes to immunodomination. These results shed light on a possible mechanism of immunodomination and highlight the need to consider multiple epitopes across the spectrum of immunogenicities in studies aimed at understanding CD8⁺ T cell immunity to viruses.     

Regulatory T Cells Prevent Th2 Immune Responses and Pulmonary Eosinophilia during Respiratory Syncytial Virus Infection in Mice

During viral infection, inflammation and recovery are tightly controlled by competing proinflammatory and regulatory immune pathways. Respiratory syncytial virus (RSV) is the leading global cause of infantile bronchiolitis, which is associated with recurrent wheeze and asthma diagnosis in later life. Th2-driven disease has been well described under some conditions for RSV-infected mice. In the present studies, we used the Foxp3^DTR mice (which allow specific conditional depletion of Foxp3⁺ T cells) to investigate the functional effects of regulatory T cells (Tregs) during A2-strain RSV infection. Infected Treg-depleted mice lost significantly more weight than wild-type mice, indicating enhanced disease. This enhancement was characterized by increased cellularity in the bronchoalveolar lavage (BAL) fluid and notable lung eosinophilia not seen in control mice. This was accompanied by abundant CD4⁺ and CD8⁺ T cells exhibiting an activated phenotype and induction of interleukin 13 (IL-13)- and GATA3-expressing Th2-type CD4⁺ T cells that remained present in the airways even 14 days after infection. Therefore, Treg cells perform vital anti-inflammatory functions during RSV infection, suppressing pathogenic T cell responses and inhibiting lung eosinophilia. These findings provide additional evidence that dysregulation of normal immune responses to viral infection may contribute to severe RSV disease.     

Systemic Toll-Like Receptor Ligation and Selective Killing of Dendritic Cell Subsets Fail To Dissect Priming Pathways for Anti-Vaccinia Virus CD8+ T Cells

CD8⁺ T cell responses can be generated by direct or cross-priming mechanisms, and several mouse models have been used to reveal which of these is the most important pathway for various viruses. Among these models is systemic treatment of mice with a CpG-containing oligodeoxynucleotide (CpG) to mature all dendritic cells (DCs), rendering them incapable of cross-presentation. A second is the use of cytochrome c (cytc) as a selective poison of the subsets of DCs able to cross-present antigen. In this study, using two vaccinia virus (VACV) strains, namely, WR and MVA, we found that the CpG and cytc methods gave conflicting data. Moreover, we show for both strains of VACV that treatment of mice with CpG and cytc inhibited CD8⁺ T cell responses to antigens designed to prime exclusively by direct presentation. Further investigation of the CpG method found that the extent to which priming is inhibited depends on the antigen examined, immunization route, replication ability of the virus, and, crucially, immunization dose. We suggest that greater caution is required when interpreting data using these methods and that priming pathways for antiviral CD8⁺ T cells are not simply separated according to DC subsets or their maturation state.     

Vaccinia virus Transmission through Experimentally Contaminated Milk Using a Murine Model

Bovine vaccinia (BV) is a zoonosis caused by Vaccinia virus (VACV), which affects dairy cattle and humans. Previous studies have detected the presence of viable virus particles in bovine milk samples naturally and experimentally contaminated with VACV. However, it is not known whether milk contaminated with VACV could be a route of viral transmission. However, anti-Orthopoxvirus antibodies were detected in humans from BV endemic areas, whom had no contact with affected cows, which suggest that other VACV transmission routes are possible, such as consumption of contaminated milk and dairy products. Therefore, it is important to study the possibility of VACV transmission by contaminated milk. This study aimed to examine VACV transmission, pathogenesis and shedding in mice orally inoculated with experimentally contaminated milk. Thirty mice were orally inoculated with milk containing 10⁷ PFU/ml of VACV, and ten mice were orally inoculated with uncontaminated milk. Clinical examinations were performed for 30 consecutive days, and fecal samples and oral swabs (OSs) were collected every other day. Mice were euthanized on predetermined days, and tissue and blood samples were collected. Nested-PCR, plaque reduction neutralization test (PRNT), viral isolation, histopathology, and immunohistochemistry (IHC) methods were performed on the collected samples. No clinical changes were observed in the animals. Viral DNA was detected in feces, blood, OSs and tissues, at least in one of the times tested. The lungs displayed moderate to severe interstitial lymphohistiocytic infiltrates, and only the heart, tonsils, tongue, and stomach did not show immunostaining at the IHC analysis. Neutralizing antibodies were detected at the 20^th and 30^th days post infection in 50% of infected mice. The results revealed that VACV contaminated milk could be a route of viral transmission in mice experimentally infected, showing systemic distribution and shedding through feces and oral mucosa, albeit without exhibiting any clinical signs.     

A primary study of the subgroups of T lymphocytes in MHV-3 induced chronic viral hepatitis

To study the contribution of T cell subsets in the pathogenesis of Murine hepatitis virus Type 3 (MHV-3) induced chronic viral hepatitis in C3H/Hej mice, ninety C3H/Hej mice were chosen to individually receive 10 plaque forming units (PFU) of MHV-3 intraperitoneally. The changes of virus titer and pathology in liver tissue were examined by standard plaque assay and by the hematoxylin/eosin (HE) staining method from 2 days post MHV-3 infection. The ratios of T cell subsets including CD3⁺CD4⁺CD8⁻, CD3⁺CD4⁻CD8⁺, CD3⁺CD4⁻CD8⁻, CD3⁺CD4⁺CD25⁺, CD3⁺CD4⁺CD25⁻ and CD3⁺ CD4⁻CD25⁺ T lymphocyte of total T lymphocytes in blood, spleen and liver were examined at 0, 2, 4, 6, 8, 10, 12, 15, 20, 25, 30, 40 days post MHV-3 infection by flow cytosorting. We observed that the virus titer raised and showed persistent virus duplications and inflammatory changes in the livers of C3H/Hej mice from 2 days post MHV-3 infection. The double negative T cell (DN Treg cell) and CD4⁺CD25⁺ T cell ratios increased significantly from 2 days post MHV-3 infection in C3H/Hej mice, and CD3⁺CD4⁺CD8⁻, CD3⁺CD4⁻CD8⁺, CD3⁺CD4⁺CD25⁻ and CD3⁺CD4⁻CD25⁺ T cell ratios decreased accordingly. In conclusion, the changes of virus titer and pathology in the livers of C3H/Hej mice post MHV-3 suggest their contribution to viral persistence. Further characterizations of DN Treg cells are that infection indicates that MHV-3 could induce the chronic inflammation in livers of C3H/Hej mice. The increase of the DN Treg cell and CD4⁺CD25⁺ T cell ratios in C3H/Hej mice post MHV-3 infection suggests that DN Treg cells and CD4⁺CD25⁺ T cells may both have important suppressive immunomodulation functions in the development of chronic viral hepatitis and have important roles in the virus persistent infection. Further characterizations of DNT cell and CD4⁺CD25⁺ T cell are under investigation. Foundation items: National Nature Science Foundation (30571643, 30672380), Major State Basic Research Development Program of China (2005CB522901, 2007CB512904)     

Plasmodium yoelii: Adverse outcome of non-lethal P. yoelii malaria during co-infection with Schistosoma mansoni in BALB/c mouse model

Plasmodium yoelii and Schistosoma mansoni co-infections were studied in female BALB/c mice aged 4-6 weeks to determine the effect of time and stage of concomitant infections on malaria disease outcome. Patent S. mansoni infection in BALB/c mice increased malaria peak parasitemia and caused death from an otherwise non-lethal, self-resolving P. yoelii malaria infection. Exacerbation of malaria parasitemia occurred during both pre-patent and patent S. mansoni infection resulting in a delay of 4-8 days in malaria parasite resolution in co-infected mice. Praziquantel administered to mice with patent schistosome infection protected from fatal outcome during co-infection. However, this treatment did not completely clear the worm infestation, nor did it reduce the peak malaria parasitemia reached, which was nonetheless resolved completely. Hepatosplenomegaly was more marked in schistosome and malaria co-infected mice compared to either infection separately. The results suggest a complex relationship between schistosome co-infection and malaria disease outcome in which the timing of malaria infection in relation to schistosome acquisition is critical to disease outcome and pathology.     

Peroxiredoxin II Regulates Effector and Secondary Memory CD8+ T Cell Responses

Reactive oxygen intermediates (ROI) generated in response to receptor stimulation play an important role in cellular responses. However, the effect of increased H₂O₂ on an antigen-specific CD8⁺ T cell response was unknown. Following T cell receptor (TCR) stimulation, the expression and oxidation of peroxiredoxin II (PrdxII), a critical antioxidant enzyme, increased in CD8⁺ T cells. Deletion of PrdxII increased ROI, S phase entry, division, and death during in vitro division. During primary acute viral and bacterial infection, the number of effector CD8⁺ T cells in PrdxII-deficient mice was increased, while the number of memory cells were similar to those of the wild-type cells. Adoptive transfer of P14 TCR transgenic cells demonstrated that the increased expansion of effector cells was T cell autonomous. After rechallenge, effector CD8⁺ T cells in mutant animals were more skewed to memory phenotype than cells from wild-type mice, resulting in a larger secondary memory CD8⁺ T cell pool. During chronic viral infection, increased antigen-specific CD8⁺ T cells accumulated in the spleens of PrdxII mutant mice, causing mortality. These results demonstrate that PrdxII controls effector CD8⁺ T cell expansion, secondary memory generation, and immunopathology.