Measles virus-induced immune suppression in the cotton rat (Sigmodon hispidus) model depends on viral glycoproteins.

Immune suppression during measles accounts for most of the morbidity and mortality associated with the virus infection. Experimental study of this phenomenon has been hampered by the lack of a suitable animal model. We have used the cotton rat to demonstrate that mitogen-induced proliferation of spleen cells from measles virus-infected animals is impaired. Proliferation inhibition is seen in all lymphocyte subsets and is not dependent on viral replication. Cells which express the viral glycoproteins (hemagglutinin and fusion protein) transiently by transfection induce proliferation inhibition after intraperitoneal inoculation, whereas application of a recombinant measles virus in which measles virus glycoproteins are replaced with the vesicular stomatitis virus G protein does not have an antiproliferative effect. Therefore, in vivo expression of measles virus glycoproteins is sufficient and necessary to induce inhibition of lymphocyte proliferation.     

Cytokine Imbalance after Measles Virus Infection Has No Correlation with Immune Suppression

Measles virus infection leads to immune suppression. A potential mechanism is the reduction of interleukin 12 (IL-12) secretion during acute measles, resulting in a TH2 response. Studies in humans have reported conflicting results, detecting either a TH2 or a TH1 response. We have investigated the correlation between a TH2 response and immune suppression in specific-pathogen-free inbred cotton rats which were infected with measles vaccine and wild-type viruses. After infection of bone marrow-derived macrophages with wild-type virus, IL-12 secretion was reduced in contrast to the level for vaccine virus infection. In bronchoalveolar lavage cells, IL-12 secretion was suppressed after infection with both wild-type and vaccine virus on days 2, 4, and 6 and was detectable on days 8 and 10. After stimulation of mediastinal lymph node and spleen cells with UV-inactivated measles virus at various time points after infection, gamma interferon but no IL-4 was found. After stimulation with phorbol myristate acetate-ionomycin, high gamma interferon and low IL-4 levels were detected. To investigate whether the secretion of IL-4 contributes to immune suppression, a recombinant vaccine virus was created which secretes cotton rat IL-4. After infection with this recombinant virus, IL-4 secretion was enhanced. However, neither inhibition of concanavalin A-stimulated spleen cells nor keyhole limpet hemocyanin-specific proliferation of spleen cells was altered after infection with the recombinant virus in comparison to the levels with the parental virus. Our data indicate that measles virus infection leads to a decrease in IL-12 secretion and an increase in IL-4 secretion, but this does not seem to correlate with immune suppression.Acute measles is caused by infection with measles virus (MV) and is associated with high morbidity and mortality. The main reason for these is thought to be immune suppression due to MV infection. Studies addressing the mechanism of immune suppression have found evidence for a number of possible mechanisms. These include unidentified soluble mediators (10, 39, 40), interference with the type I interferon (IFN) system (for a review, see reference 9), apoptosis (8), impaired lymphoproliferation (23, 35), interleukin 12 (IL-12) downregulation (1, 18), and impaired dendritic cell (DC) function (36). Some of the mechanisms may operate in conjunction with each other; e.g., it is possible that the downregulation of IL-12 might lead to the development of a T-helper 2 (TH2) response that results in impaired lymphoproliferation. The evidence for this line of argument is that human macrophages, after stimulation through Toll-like receptor 4 (TLR-4), secrete less IL-12 when infected with MV (18). In MV-infected humans, the percentage of IL-12-expressing macrophages is reduced (1), and in Rhesus macaques, lowered serum levels of IL-12 have been found during MV infection (31). In macaques, this correlates with an increase of eosinophils in peripheral blood, indicative of increased secretion of IL-5 (31).In serum or supernatants of peripheral blood lymphocytes (PBLs) stimulated with phorbol myristate acetate (PMA)-ionomycin from patients with acute measles, an upregulation of IFN-γ and IL-2 (12) (or IL-2-receptor [37]) was found before and during the rash, whereas after the rash, a reduction in IL-2 and an increase in IL-4 (12) or IL-10 (22) levels were observed. In contrast, other studies found normal levels of IFN-γ and IL-2 secreted by stimulated PBLs (42) and increased levels of IFN-γ after the rash secreted by PMA-ionomycin-stimulated CD8 T cells (22).Results from the analysis of cytokines secreted by lymphocytes from MV vaccinees were also conflicting. In one study, an upregulation of IL-4 and downregulation of IFN-γ were found in the supernatants of phytohemagglutinin-stimulated cells (41). In another study, increased IL-12 and IFN-γ levels were detected after stimulation with MV antigen (11). In a third study, no differences in the expression levels of IL-4, IL-5, or IL-12 were found between vaccinated and unvaccinated individuals by ex vivo hybridization for cytokine mRNA in PBLs (21). However, when PBLs from vaccinees were stimulated with MV and analyzed on a per-cell basis by limiting dilution, it was found that nearly all T cells expressed IFN-γ, either alone or in combination with IL-4 (16). In a similar cohort of vaccinees, it could demonstrated by a IL-4-receptor-blocking enzyme-linked immunosorbent assay (ELISA) that PBLs from about half of the individuals secreted IL-4 (7) (IFN-γ secretion was not addressed). This might be explained by a recent report suggesting that the secretion of IFN-γ and IL-4 after MV stimulation is linked to the haplotype of human major histocompatibility complex class I (MHC-I) molecules (HLA) (28).Based on these data, no clear correlation can be drawn between reduced levels of IL-12, the secretion of TH2-type cytokines, and the inhibition of lymphocyte proliferation. Furthermore, at least three variables seem to confound the analysis: (i) the use of samples from patients with acute measles (wild-type virus) versus vaccinees (vaccine virus), (ii) the method of stimulation of cells and cytokine detection, and (iii) the individual genetic makeups of the study participants.In this report, we have used inbred cotton rats to compare the abilities of wild-type and vaccine MVs to inhibit IL-12 secretion, and we have compared the secretion of TH1 and -2 cytokines after stimulation with MV antigen versus that with PMA-ionomycin. In addition, we tested a recombinant MV expressing cotton rat IL-4 for its ability to suppress the immune system.     

Neonatal immunization with a Sindbis virus-DNA measles vaccine induces adult-like neutralizing antibodies and cell-mediated immunity in the presence of maternal antibodies

Infants younger than age 9 mo do not respond reliably to the live attenuated measles vaccine due the immaturity of their immune system and the presence of maternal Abs that interfere with successful immunization. We evaluated the immune responses elicited by Sindbis virus replicon-based DNA vaccines encoding measles virus (MV) hemagglutinin (H, pMSIN-H) or both hemagglutinin and fusion (F, pMSINH-FdU) glycoproteins in neonatal mice born to naive and measles-immune mothers. Despite the presence of high levels of maternal Abs, neonatal immunization with pMSIN-H induced long-lasting, high-avidity MV plaque reduction neutralization (PRN) Abs, mainly IgG2a, that also inhibited syncytium formation in CD150(+) B95-8 cells. IgG secreting plasma cells were detected in spleen and bone marrow. Newborns vaccinated with pMSINH-FdU elicited PRN titers that surpassed the protective level (200 mIU/ml) but were short-lived, had low syncytium inhibition capacity, and lacked avidity maturation. This vaccine failed to induce significant PRN titers in the presence of placentally transferred Abs. Both pMSIN-H and pMSINH-FdU elicited strong Th1 type cell-mediated immunity, measured by T cell proliferation and IFN-gamma production, that was unaffected by maternal Abs. Newborns responded to measles DNA vaccines with similar or even higher PRN titers and cell-mediated immunity than adult mice. This study is the first demonstration that a Sindbis virus-based measles DNA vaccine can elicit robust MV immunity in neonates bypassing maternal Abs. Such a vaccine could be followed by the current live attenuated MV vaccine in a heterologous prime-boost to protect against measles early in life.     

Role of CD8(+) lymphocytes in control and clearance of measles virus infection of rhesus monkeys

The creation of an improved vaccine for global measles control will require an understanding of the immune mechanisms of measles virus containment. To assess the role of CD8(+) cytotoxic T lymphocytes in measles virus clearance, rhesus monkeys were depleted of CD8(+) lymphocytes by monoclonal anti-CD8 antibody infusion and challenged with wild-type measles virus. The CD8(+) lymphocyte-depleted animals exhibited a more extensive rash, higher viral loads at the peak of virus replication, and a longer duration of viremia than did the control antibody-treated animals. These findings indicate a central role for CD8(+) lymphocytes in the control of measles virus infections and the importance of eliciting a cell-mediated immune response in new measles vaccine strategies.     

Distinct cellular immune responses following primary and secondary influenza virus challenge in cotton rats

To evaluate cell-mediated immunity in influenza-infected cotton rats, we compared the cellular composition of spleen, mediastinal lymph nodes (MLN) and bronchoalveolar lavage (BAL) after primary and secondary infection. There was an increase in cellularity in the MLN after primary infection that was further expanded upon rechallenge. CD4(+) T cells expanded after primary infection, but there was preferential increase in the number of CD4-negative T cells following secondary challenge. After primary infection, a large proportion of the monocytes and NK cells were present in the BAL while a T cell population dominated after secondary infection. CD4(+) T cells were predominant in this population unless the animals had been challenged with heterosubtypic influenza A virus. These studies are the first to show evidence of a memory T cell response to influenza infection in cotton rats and show quantitative and qualitative differences between the recall response to homosubtypic and heterosubtypic viruses.     

Measles virus infection of thymic epithelium in the SCID-hu mouse leads to thymocyte apoptosis.

Mortality from measles is caused mostly by secondary infections associated with the depression of cellular immunity. The mechanism of immune suppression and the role of virus strain differences on the immune system are incompletely understood. SCID-hu mice were used to determine the effects of virulent, wild-type (Chicago-1) and avirulent, vaccine (Moraten) strains of measles virus (MV) on the human thymus in vivo. Chicago-1 replicated rapidly, with a 100-fold decrease in numbers of thymocytes, whereas Moraten replicated slowly, without significant thymocyte death. Productive MV infection occurred not in thymocytes but in thymic epithelial and myelomonocytic cells. Wild-type MV infection of thymic stromata leads to induction of thymocyte apoptosis and may contribute to a long-term alteration of immune responses. The extent of thymic disruption reflects the virulence of the virus, and therefore the SCID-hu mouse may serve as the first small animal model for the study of MV pathogenesis.     

Canine distemper virus epithelial cell infection is required for clinical disease but not for immunosuppression

To characterize the importance of infection of epithelial cells for morbillivirus pathogenesis, we took advantage of the severe disease caused by canine distemper virus (CDV) in ferrets. To obtain a CDV that was unable to enter epithelial cells but retained the ability to enter immune cells, we transferred to its attachment (H) protein two mutations shown to interfere with the interaction of measles virus H with its epithelial receptor, human nectin-4. As expected for an epithelial receptor (EpR)-blind CDV, this virus infected dog and ferret epithelial cells inefficiently and did not cause cell fusion or syncytium formation. On the other hand, the EpR-blind CDV replicated in cells expressing canine signaling lymphocyte activation molecule (SLAM), the morbillivirus immune cell receptor, with similar kinetics to those of wild-type CDV. While ferrets infected with wild-type CDV died within 12 days after infection, after developing severe rash and fever, animals infected with the EpR-blind virus showed no clinical signs of disease. Nevertheless, both viruses spread rapidly and efficiently in immune cells, causing similar levels of leukopenia and inhibition of lymphocyte proliferation activity, two indicators of morbillivirus immunosuppression. Infection was documented for airway epithelia of ferrets infected with wild-type CDV but not for those of animals infected with the EpR-blind virus, and only animals infected with wild-type CDV shed virus. Thus, epithelial cell infection is necessary for clinical disease and efficient virus shedding but not for immunosuppression.     

Modulation of Immune System Function by Measles Virus Infection: Role of Soluble Factor and Direct Infection

Measles virus infection can result in a variety of immunologic defects. We have begun studies to determine the basis for the lack of immune responsiveness to antigen and mitogen following infection. Here we present data showing that Epstein-Barr virus-transformed B-cell lines infected with measles virus produce a soluble factor that can inhibit antigen-specific T-cell proliferation and inhibit the proliferation of uninfected B cells. The soluble factor was neither interleukin-10, transforming growth factor β, nor alpha/beta interferon. B cells infected with measles virus or treated with the soluble factor were unable to present antigen to T cells in a manner that supported antigen-specific proliferation. This could represent one mechanism of how measles virus limits T-cell expansion. However, we found that once CD4⁺ or CD8⁺ T cells were activated, their cytolytic activity was intact whether infected with measles virus or treated with soluble factor. Thus, while slow to be generated these cytoxic cells could participate in viral clearance.     

Evasion of host defenses by measles virus: wild-type measles virus infection interferes with induction of Alpha/Beta interferon production

Measles is a highly contagious disease currently responsible for over one million childhood deaths, particularly in the developing world. Since alpha/beta interferons (IFNs) are pivotal players both in nonspecific antiviral immunity and in specific cellular responses, their induction or suppression by measles virus (MV) could influence the outcome of a viral infection. In this study we compare the IFN induction and sensitivity of laboratory-passaged attenuated MV strains Edmonston and Moraten with those of recent wild-type viruses isolated and passaged solely on human peripheral blood mononuclear cells (PBMC) or on the B958 marmoset B-cell line. We report that two PBMC-grown wild-type measles isolates and two B958-grown strains of MV induce 10- to 80-fold-lower production of IFN by phytohemagglutinin-stimulated peripheral blood lymphocytes (PBL) compared to Edmonston and Moraten strains of measles. Preinfection of PBL with these non-IFN-inducing MV isolates prevents Edmonston-induced but not double-stranded-RNA-induced IFN production. This suggests that the wild-type viruses can actively inhibit Edmonston-induced IFN synthesis and that this is not occurring by double-stranded RNA. Furthermore, the wild-type MV is more sensitive than Edmonston MV to the effect of IFN. MV is thus able to suppress the synthesis of the earliest mediator of antiviral immunity, IFN-alpha/beta. This could have important implications in the virulence and spread of MV.     

Immature CD4+CD8+ Thymocytes Are Preferentially Infected by Measles Virus in Human Thymic Organ Cultures

Cells of the human immune system are important target cells for measles virus (MeV) infection and infection of these cells may contribute to the immunologic abnormalities and immune suppression that characterize measles. The thymus is the site for production of naïve T lymphocytes and is infected during measles. To determine which populations of thymocytes are susceptible to MeV infection and whether strains of MeV differ in their ability to infect thymocytes, we used ex vivo human thymus organ cultures to assess the relative susceptibility of different subpopulations of thymocytes to infection with wild type and vaccine strains of MeV. Thymocytes were susceptible to MeV infection with the most replication in immature CD4⁺CD8⁺ double positive cells. Susceptibility correlated with the level of expression of the MeV receptor CD150. Wild type strains of MeV infected thymocytes more efficiently than the Edmonston vaccine strain. Thymus cultures from children ≥3 years of age were less susceptible to MeV infection than cultures from children 5 to 15 months of age. Resistance in one 7 year-old child was associated with production of interferon-gamma suggesting that vaccination may result in MeV-specific memory T cells in the thymus. We conclude that immature thymocytes are susceptible to MeV infection and thymocyte infection may contribute to the immunologic abnormalities associated with measles.     

Immune responses in pigs vaccinated with adjuvanted and non-adjuvanted A(H1N1)pdm/09 influenza vaccines used in human immunization programmes

Following the emergence and global spread of a novel H1N1 influenza virus in 2009, two A(H1N1)pdm/09 influenza vaccines produced from the A/California/07/09 H1N1 strain were selected and used for the national immunisation programme in the United Kingdom: an adjuvanted split virion vaccine and a non-adjuvanted whole virion vaccine. In this study, we assessed the immune responses generated in inbred large white pigs (Babraham line) following vaccination with these vaccines and after challenge with A(H1N1)pdm/09 virus three months post-vaccination. Both vaccines elicited strong antibody responses, which included high levels of influenza-specific IgG1 and haemagglutination inhibition titres to H1 virus. Immunisation with the adjuvanted split vaccine induced significantly higher interferon gamma production, increased frequency of interferon gamma-producing cells and proliferation of CD4(-)CD8(+) (cytotoxic) and CD4(+)CD8(+) (helper) T cells, after in vitro re-stimulation. Despite significant differences in the magnitude and breadth of immune responses in the two vaccinated and mock treated groups, similar quantities of viral RNA were detected from the nasal cavity in all pigs after live virus challenge. The present study provides support for the use of the pig as a valid experimental model for influenza infections in humans, including the assessment of protective efficacy of therapeutic interventions.     

The Vaccinia virus complement control protein modulates adaptive immune responses during infection

Complement activation is an important component of the innate immune response against viral infection and also shapes adaptive immune responses. Despite compelling evidence that complement activation enhances T cell and antibody (Ab) responses during viral infection, it is unknown whether inhibition of complement by pathogens alters these responses. Vaccinia virus (VACV) modulates complement activation by encoding a complement regulatory protein called the vaccinia virus complement control protein (VCP). Although VCP has been described as a virulence factor, the mechanisms by which VCP enhances VACV pathogenesis have not been fully defined. Since complement is necessary for optimal adaptive immune responses to several viruses, we hypothesized that VCP contributes to pathogenesis by modulating anti-VACV T cell and Ab responses. In this study, we used an intradermal model of VACV infection to compare pathogenesis of wild-type virus (vv-VCPwt) and a virus lacking VCP (vv-VCPko). vv-VCPko formed smaller lesions in wild-type mice but not in complement-deficient mice. Attenuation of vv-VCPko correlated with increased accumulation of T cells at the site of infection, enhanced neutralizing antibody responses, and reduced viral titers. Importantly, depleting CD8(+) T cells together with CD4(+) T cells, which also eliminated T helper cell-dependent Ab responses, restored vv-VCPko to wild-type levels of virulence. These results suggest that VCP contributes to virulence by dampening both antibody and T cell responses. This work provides insight into how modulation of complement by poxviruses contributes to virulence and demonstrates that a pathogen-encoded complement regulatory protein can modulate adaptive immunity.     

Avoiding the void: cell-to-cell spread of human viruses

The initial stages of animal virus infection are generally described as the binding of free virions to permissive target cells followed by entry and replication. Although this route of infection is undoubtedly important, many viruses that are pathogenic for humans, including HIV-1, herpes simplex virus and measles, can also move between cells without diffusing through the extracellular environment. Cell-to-cell spread not only facilitates rapid viral dissemination, but may also promote immune evasion and influence disease. This Review discusses the various mechanisms by which viruses move directly between cells and the implications of this for viral dissemination and pathogenesis.     

A Recombinant Measles Vaccine Virus Expressing Wild-Type Glycoproteins: Consequences for Viral Spread and Cell Tropism

Wild-type, lymphotropic strains of measles virus (MV) and tissue culture-adapted MV vaccine strains possess different cell tropisms. This observation has led to attempts to identify the viral receptors and to characterize the functions of the MV glycoproteins. We have functionally analyzed the interactions of MV hemagglutinin (H) and fusion (F) proteins of vaccine (Edmonston) and wild-type (WTF) strains in different combinations in transfected cells. Cell-cell fusion occurs when both Edmonston F and H proteins are expressed in HeLa or Vero cells. The expression of WTF glycoproteins in HeLa cells did not result in syncytia, yet they fused efficiently with cells of lymphocytic origin. To further investigate the role of the MV glycoproteins in virus cell entry and also the role of other viral proteins in cell tropism, we generated recombinant vaccine MVs containing one or both glycoproteins from WTF. These viruses were viable and grew similarly in lymphocytic cells. Recombinant viruses expressing the WTFH protein showed a restricted spread in HeLa cells but spread efficiently in Vero cells. Parental WTF remained restricted in both cell types. Therefore, not only differential receptor usage but also other cell-specific factors are important in determining MV cell tropism.     

Hemagglutinin protein of wild-type measles virus activates toll-like receptor 2 signaling

Pattern recognition via Toll-like receptors (TLR) by antigen-presenting cells is an important element of innate immunity. We report that wild-type measles virus but not vaccine strains activate cells via both human and murine TLR2, and this is a property of the hemagglutinin (H) protein. The ability to activate cells via TLR2 by wild-type MV H protein is abolished by mutation of a single amino acid, asparagine at position 481 to tyrosine, as is found in attenuated strains, which is important for interaction with CD46, the receptor for these strains. TLR2 activation by MV wild-type H protein stimulates induction of proinflammatory cytokines such as interleukin-6 (IL-6) in human monocytic cells and surface expression of CD150, the receptor for all MV strains. Confirming the specificity of this interaction, wild-type H protein did not induce IL-6 release in macrophages from TLR2-/- mice. Thus, the unique property of MV wild-type strains to activate TLR2-dependent signals might essentially contribute not only to immune activation but also to viral spread and pathogenicity by upregulating the MV receptor on monocytes.     

Measles viruses possessing the polymerase protein genes of the Edmonston vaccine strain exhibit attenuated gene expression and growth in cultured cells and SLAM knock-in mice

Live attenuated vaccines against measles have been developed through adaptation of clinical isolates of measles virus (MV) in various cultured cells. Analyses using recombinant MVs with chimeric genomes between wild-type and Edmonston vaccine strains indicated that viruses possessing the polymerase protein genes of the Edmonston strain exhibited attenuated viral gene expression and growth in cultured cells as well as in mice expressing an MV receptor, signaling lymphocyte activation molecule, regardless of whether the virus genome had the wild-type or vaccine-type promoter sequence. These data demonstrate that the polymerase protein genes of the Edmonston strain contribute to its attenuated phenotype.     

Measles Immune Suppression: Lessons from the Macaque Model

Measles remains a significant childhood disease, and is associated with a transient immune suppression. Paradoxically, measles virus (MV) infection also induces robust MV-specific immune responses. Current hypotheses for the mechanism underlying measles immune suppression focus on functional impairment of lymphocytes or antigen-presenting cells, caused by infection with or exposure to MV. We have generated stable recombinant MVs that express enhanced green fluorescent protein, and remain virulent in non-human primates. By performing a comprehensive study of virological, immunological, hematological and histopathological observations made in animals euthanized at different time points after MV infection, we developed a model explaining measles immune suppression which fits with the “measles paradox”. Here we show that MV preferentially infects CD45RA⁻ memory T-lymphocytes and follicular B-lymphocytes, resulting in high infection levels in these populations. After the peak of viremia MV-infected lymphocytes were cleared within days, followed by immune activation and lymph node enlargement. During this period tuberculin-specific T-lymphocyte responses disappeared, whilst strong MV-specific T-lymphocyte responses emerged. Histopathological analysis of lymphoid tissues showed lymphocyte depletion in the B- and T-cell areas in the absence of apoptotic cells, paralleled by infiltration of T-lymphocytes into B-cell follicles and reappearance of proliferating cells. Our findings indicate an immune-mediated clearance of MV-infected CD45RA⁻ memory T-lymphocytes and follicular B-lymphocytes, which causes temporary immunological amnesia. The rapid oligoclonal expansion of MV-specific lymphocytes and bystander cells masks this depletion, explaining the short duration of measles lymphopenia yet long duration of immune suppression.     

Attenuated Salmonella enterica serovar Typhi and Shigella flexneri 2a strains mucosally deliver DNA vaccines encoding measles virus hemagglutinin,inducing specific immune responses and protection in cotton rats

Measles remains a leading cause of child mortality in developing countries. Residual maternal measles antibodies and immunologic immaturity dampen immunogenicity of the current vaccine in young infants. Because cotton rat respiratory tract is susceptible to measles virus (MV) replication after intranasal (i.n.) challenge, this model can be used to assess the efficacy of MV vaccines. Pursuing a new measles vaccine strategy that might be effective in young infants, we used attenuated Salmonella enterica serovar Typhi CVD 908-htrA and Shigella flexneri 2a CVD 1208 vaccines to deliver mucosally to cotton rats eukaryotic expression plasmid pGA3-mH and Sindbis virus-based DNA replicon pMSIN-H encoding MV hemagglutinin (H). The initial i.n. dose-response with bacterial vectors alone identified a well-tolerated dosage (1 x 10(9) to 7 x 10(9) CFU) and a volume (20 micro l) that elicited strong antivector immune responses. Animals immunized i.n. on days 0, 28, and 76 with bacterial vectors carrying DNA plasmids encoding MV H or immunized parenterally with these naked DNA vaccine plasmids developed MV plaque reduction neutralizing antibodies and proliferative responses against MV antigens. In a subsequent experiment of identical design, cotton rats were challenged with wild-type MV 1 month after the third dose of vaccine or placebo. MV titers were significantly reduced in lung tissue of animals immunized with MV DNA vaccines delivered either via bacterial live vectors or parenterally. Since attenuated serovar Typhi and S. flexneri can deliver measles DNA vaccines mucosally in cotton rats, inducing measles immune responses (including neutralizing antibodies) and protection, boosting strategies can now be evaluated in animals primed with MV DNA vaccines.