Teresa R. Johnson

The Role of IFN in Respiratory Syncytial Virus Pathogenesis

Formalin-inactivated respiratory syncytial virus (RSV) vaccine preparations have been shown to cause enhanced disease in naive hosts following natural infection. In this study we demonstrate a similar pattern of enhanced disease severity following primary RSV infection of IFN-nonresponsive STAT1−/− mice. STAT1−/− mice showed markedly increased illness compared with wild-type BALB/c animals following RSV inoculation despite similar lung virus titers and rates of virus clearance. Histologically, STAT1−/− animals had eosinophilic and neutrophilic pulmonary infiltrates not present in wild-type or IFN-γ−/−-infected mice. In cytokine analyses of infected lung tissue, IFN-γ was induced in both STAT1−/− and wild-type mice, with preferential IL-4, IL-5, and IL-13 induction only in the STAT1−/− animals. Eotaxin was detected in the lungs of both wild-type and STAT1−/− mice following infection, with a 1.7-fold increase over wild-type in the STAT1−/− mice. Using a peptide epitope newly identified in the RSV fusion protein, we were able to demonstrate that wild-type memory CD4+ T cells stimulated by this peptide produce primarily IFN-γ, while STAT1−/−CD4+ cells produce primarily IL-13. These findings suggest that STAT1 activation by both type I (αβ) and type II (γ) IFNs plays an important role in establishing a protective, Th1 Ag-specific immune response to RSV infection.

IL-13 is sufficient for respiratory syncytial virus G glycoprotein-induced eosinophilia after respiratory syncytial virus challenge

Although well studied in settings of helminth infection and allergen sensitization, the combined contributions of IL-4 and IL-13 and their signaling pathways in models of viral pathogenesis have not been reported. Using a murine model of respiratory syncytial virus (RSV) infection, we evaluated the contribution of IL-13, alone and in conjunction with IL-4, during immunization with recombinant vaccinia virus expressing RSV G glycoprotein (vvGs) or with formalin-inactivated RSV (FI-RSV). We showed that both IL-4 and IL-13 activity must be inhibited to modulate G-specific responses resulting in severe RSV-induced disease. Inhibition of IL-4 or IL-13 activity alone had minimal impact on disease in vvGs-immunized mice. However, treatment of IL-4-deficient mice with IL-13Ra during vvGs immunization reduced IL-5, IL-13, and eotaxin production and pulmonary eosinophilia after RSV challenge. In contrast, FI-RSV-induced immune responses were diminished when either IL-4 or IL-13 activity was blocked. After RSV challenge, these type 2 T cell responses were also diminished in vvGs-primed IL-4Rα-deficient mice. Our data suggest that secreted vvGs uses mechanisms requiring signaling through the IL-4Rα-chain by either IL-4 or IL-13 for induction of eosinophilia and is the first description of the relative contributions of IL-4, IL-13, and their receptors in viral pathogenesis.

Respiratory Syncytial Virus Glycoprotein G Interacts with DC-SIGN and L-SIGN To Activate ERK1 and ERK2

ABSTRACT Respiratory syncytial virus (RSV) interaction with epithelial and dendritic cells (DCs) is known to require divalent cations, suggesting involvement of C-type lectins. RSV infection and maturation of primary human DCs are reduced in a dose-dependent manner by EDTA. Therefore, we asked whether RSV infection involves DC-SIGN (CD209) or its isoform L-SIGN (CD299) (DC-SIGN/R). Using surface plasmon resonance analysis, we demonstrated that the attachment G glycoprotein of RSV binds both DC- and L-SIGN. However, neutralization of DC- and L-SIGN on primary human DCs did not inhibit RSV infection, demonstrating that interactions between RSV G and DC- or L-SIGN are not required for productive infection. Thus, neither DC- nor L-SIGN represents a functional receptor for RSV. However, inhibition of these interactions increased DC activation, as evidenced by significantly higher levels of alpha interferon (IFN-α), MIP-1α, and MIP-1β in plasmacytoid DCs (pDCs) exposed to RSV after neutralization of DC-and L-SIGN. To understand the molecular interactions involved, intracellular signaling events triggered by purified RSV G glycoprotein were examined in DC- and L-SIGN-transfected 3T3 cells. RSV G interaction with DC- or L-SIGN was shown to stimulate ERK1 and ERK2 phosphorylation, with statistically significant increases relative to mock-infected cells. Neutralization of DC- and L-SIGN reduced ERK1/2 phosphorylation. With increased DC activation following DC- and L-SIGN neutralization and RSV exposure, these data demonstrate that the signaling events mediated by RSV G interactions with DC/L-SIGN are immunomodulatory and diminish DC activation, which may limit induction of RSV-specific immunity.

Role for Innate IFNs in Determining Respiratory Syncytial Virus Immunopathology

Respiratory syncytial virus (RSV) is the major cause of severe lower airway disease in infants and young children, but no safe and effective RSV vaccine is yet available. The difficulties involved in RSV vaccine development were recognized in an early vaccine trial, when children immunized with a formalin-inactivated virus preparation experienced enhanced illness after natural infection. Subsequent research in animal models has shown that the vaccine-enhanced disease is mediated at least in part by memory cells producing Th2 cytokines. Previously we had observed enhanced, eosinophilic lung pathology during primary infection of IFN-deficient STAT1−/− mice that are incapable of generating Th1 CD4+ cells. To determine whether these effects depended only on Th2 cytokine secretion or involved other aspects of IFN signaling, we infected a series of 129SvEv knockout mice lacking the IFN-αβR (IFN-αβR−/−), the IFN-γR (IFN-γR−/−), or both receptors (IFN-αβγR−/−). Although both the IFN-γR−/− and the IFN-αβγR−/− animals generated strong Th2 responses to RSV-F protein epitopes, predominantly eosinophilic lung disease was limited to mice lacking both IFNRs. Although the absolute numbers of eosinophils in BAL fluids were similar between the strains, very few CD8+ T cells could be detected in lungs of IFN-αβγR−/− animals, leaving eosinophils as the predominant leukocyte. Thus, although CD4+ Th2 cell differentiation is necessary for the development of allergic-type inflammation after infection and appears to be unaffected by type I IFNs, innate IFNs clearly have an important role in determining the nature and severity of RSV disease.

Primary human mDC1, mDC2, and pDC dendritic cells are differentially infected and activated by respiratory syncytial virus.

Respiratory syncytial virus (RSV) causes recurrent infections throughout life. Vaccine development may depend upon understanding the molecular basis for induction of ineffective immunity. Because dendritic cells (DCs) are critically involved in early responses to infection, their interaction with RSV may determine the immunological outcome of RSV infection. Therefore, we investigated the ability of RSV to infect and activate primary mDCs and pDCs using recombinant RSV expressing green fluorescent protein (GFP). At a multiplicity of infection of 5, initial studies demonstrated ∼6.8% of mDC1 and ∼0.9% pDCs were infected. We extended these studies to include CD1c−CD141+ mDC2, finding mDC2 infected at similar frequencies as mDC1. Both infected and uninfected cells upregulated phenotypic markers of maturation. Divalent cations were required for infection and maturation, but maturation did not require viral replication. There is evidence that attachment and entry/replication processes exert distinct effects on DC activation. Cell-specific patterns of RSV-induced maturation and cytokine production were detected in mDC1, mDC2, and pDC. We also demonstrate for the first time that RSV induces significant TIMP-2 production in all DC subsets. Defining the influence of RSV on the function of selected DC subsets may improve the likelihood of achieving protective vaccine-induced immunity.

TLR9 agonist, but not TLR7/8, functions as an adjuvant to diminish FI-RSV vaccine-enhanced disease, while either agonist used as therapy during primary RSV infection increases disease severity

Agonists for TLR7, TLR8, and TLR9 have been shown to enhance vaccine immunogenicity. We evaluated the impact of TLR activation on RSV disease in a murine model by administering TLR7/8 and TLR9 agonists during FI-RSV immunization or RSV infection. CpG administered during immunization reduced disease following challenge as evidenced by decreased lung pathology, illness, and cytokines. In marked contrast, TLR7/8 agonist had little impact. To evaluate potential therapeutic use, TLR agonists were administered during primary infection. Although type 2 cytokine responses decreased and type 1 cytokines and MIP-1-alpha/beta increased, both TLR7/8 and TLR9 agonists increased clinical symptoms and pulmonary inflammation when administered during primary infection. Thus, TLR9-induced signaling during FI-RSV immunization reduced vaccine-enhanced disease whereas immunostimulatory properties of TLR agonists enhanced disease severity when used during RSV infection. Immunomodulation elicited by TLR9 agonist confirms the adjuvant potential of TLR agonists during RSV immunization. However, in contrast to work done with HIV-1 vaccines, the inability of TLR7/8 agonist to boost type 1 vaccine-induced RSV immunity demonstrates pathogen-TLR specificity. These data reveal that the timing of administration of immunomodulatory agents is critical. Furthermore, these data underscore that amplification of anti-viral immune responses may result in immunopathology rather than immune-mediated protection.

Modified vaccinia virus Ankara immunization protects against lethal challenge with recombinant vaccinia virus expressing murine interleukin-4.

ABSTRACT Recent events have raised concern over the use of pathogens, including variola virus, as biological weapons. Vaccination with Dryvax is associated with serious side effects and is contraindicated for many people, and the development of a safer effective smallpox vaccine is necessary. We evaluated an attenuated vaccinia virus, modified vaccinia virus Ankara (MVA), by use of a murine model to determine its efficacy against an intradermal (i.d.) or intranasal (i.n.) challenge with vaccinia virus (vSC8) or a recombinant vaccinia virus expressing murine interleukin-4 that exhibits enhanced virulence (vSC8-mIL4). After an i.d. challenge, 15 of 16 mice who were inoculated with phosphate-buffered saline developed lesions, one dose of intramuscularly administered MVA was partially protective (3 of 16 mice developed lesions), and the administration of two or three doses of MVA was completely protective (0 of 16 mice developed lesions). In unimmunized mice, an i.n. challenge with vSC8 caused a significant but self-limited illness, while vSC8-mIL4 resulted in lethal infections. Immunization with one or two doses of MVA prevented illness and reduced virus titers in mice who were challenged with either vSC8 or vSC8-mIL4. MVA induced a dose-related neutralizing antibody and vaccinia virus-specific CD8+-T-cell response. Mice immunized with MVA were fully protected from a low-dose vSC8-mIL4 challenge despite a depletion of CD4+ cells, CD8+ cells, or both T-cell subsets or an antibody deficiency. CD4+- or CD8+-T-cell depletion reduced the protection against a high-dose vSC8-mIL4 challenge, and the depletion of both T-cell subsets was associated with severe illness and higher vaccinia virus titers. Thus, MVA induces broad humoral and cellular immune responses that can independently protect against a molecularly modified lethal poxvirus challenge in mice. These data support the continued development of MVA as an alternative candidate vaccine for smallpox.

Pulmonary eosinophilia requires interleukin-5, eotaxin-1, and CD4+ T cells in mice immunized with respiratory syncytial virus G glycoprotein

Severe illness, type 2 cytokine production, and pulmonary eosinophilia are adverse immune responses resulting from respiratory syncytial virus (RSV) challenge of vvGs‐immunized mice. We have shown IL‐4 and IL‐13 activity must be simultaneously inhibited to reduce disease severity. We now address the contributions of IL‐5, eotaxin‐1, and CD4+ and CD8+ T cells to the induction of disease‐enhancing immune responses. Depletion of CD4+ T cells during immunization prevented IL‐4, IL‐13, and eotaxin‐1 production, diminished eosinophilia, and reduced weight loss. Conversely, CD8+ T cell depletion did not decrease eosinophilia, weight loss, or type 2 cytokines but did dramatically reduce mucus production and increase eotaxin production. Anti‐IL‐5 administration at immunization or challenge significantly decreased pulmonary eosinophilia. Strikingly, there were not concomitant decreases in weight loss. Following RSV challenge eotaxin‐1‐deficient mice immunized with vvGs exhibited significantly less eosinophilia without decreased weight loss or type 2 cytokine production. We conclude CD4+ T cell production of IL‐5 and induction of eotaxin‐1 are required for vvGs‐induced eosinophilia following RSV challenge, while CD8+ T cells appear to down‐regulate eotaxin‐1 and mucus production. In summary, we demonstrate that pulmonary eosinophilia 1) is a by‐product of memory CD4+ T cell activation, 2) does not necessarily correlate with mucus production, and, most importantly, 3) is not required for the RSV G‐induced illness in mice. These findings have important implications for the evaluation of candidate RSV vaccines.

Vaccination with Human Papillomavirus Pseudovirus-Encapsidated Plasmids Targeted to Skin Using Microneedles

Human papilloma virus-like particles (HPV VLP) serve as the basis of the current licensed vaccines for HPV. We have previously shown that encapsidation of DNA expressing the model antigen M/M2 from respiratory syncytial virus (RSV) in HPV pseudovirions (PsV) is immunogenic when delivered intravaginally. Because the HPV capsids confer tropism for basal epithelium, they represent attractive carriers for vaccination targeted to the skin using microneedles. In this study we asked: 1) whether HPV16 VLP administered by microneedles could induce protective immune responses to HPV16 and 2) whether HPV16 PsV-encapsidated plasmids delivered by microneedles could elicit immune responses to both HPV and the antigen delivered by the transgene. Mice immunized with HPV16 VLP coated microneedles generated robust neutralizing antibody responses and were protected from HPV16 challenge. Microneedle arrays coated with HPV16-M/M2 or HPV16-F protein (genes of RSV) were then tested and dose-dependent HPV and F-specific antibody responses were detected post-immunization, and M/M2-specific T-cell responses were detected post RSV challenge, respectively. HPV16 PsV-F immunized mice were fully protected from challenge with HPV16 PsV and had reduced RSV viral load in lung and nose upon intranasal RSV challenge. In summary, HPV16 PsV-encapsidated DNA delivered by microneedles induced neutralizing antibody responses against HPV and primed for antibody and T-cell responses to RSV antigens encoded by the encapsidated plasmids. Although the immunogenicity of the DNA component was just above the dose response threshold, the HPV-specific immunity was robust. Taken together, these data suggest microneedle delivery of lyophilized HPV PsV could provide a practical, thermostable combined vaccine approach that could be developed for clinical evaluation.

Coadministration of Polyinosinic:Polycytidylic Acid and Immunostimulatory Complexes Modifies Antigen Processing in Dendritic Cell Subsets and Enhances HIV Gag-Specific T Cell Immunity

Currently approved adjuvants induce protective Ab responses but are more limited for generating cellular immunity. In this study, we assessed the effect of combining two adjuvants with distinct mechanisms of action on their ability to prime T cells: the TLR3 ligand, polyinosinic:polycytidylic acid (poly I:C), and immunostimulatory complexes (ISCOMs). Each adjuvant was administered alone or together with HIV Gag protein (Gag), and the magnitude, quality, and phenotype of Gag-specific T cell responses were assessed. For CD8 T cells, all adjuvants induced a comparable response magnitude, but combining poly I:C with ISCOMs induced a high frequency of CD127+, IL-2–producing cells with decreased expression of Tbet compared with either adjuvant alone. For CD4 T cells, combining poly I:C and ISCOMs increased the frequency of multifunctional cells, producing IFN-γ, IL-2, and TNF, and the total magnitude of the response compared with either adjuvant alone. CD8 or CD4 T cell responses induced by both adjuvants mediated protection against Gag-expressing Listeria monocytogenes or vaccinia viral infections. Poly I:C and ISCOMs can alter Ag uptake and/or processing, and we therefore used fluorescently labeled HIV Gag and DQ-OVA to assess these mechanisms, respectively, in multiple dendritic cell subsets. Poly I:C promoted uptake and retention of Ag, whereas ISCOMs enhanced Ag degradation. Combining poly I:C and ISCOMs caused substantial death of dendritic cells but persistence of degraded Ag. These data illustrate how combining adjuvants, such as poly I:C and ISCOMs, that modulate Ag processing and have potent innate activity, can enhance the magnitude, quality, and phenotype of T cell immunity.