A recombinant herpes virus expressing influenza hemagglutinin confers protection and induces antibody-dependent cellular cytotoxicity.

Despite widespread yearly vaccination, influenza leads to significant morbidity and mortality across the globe. To make a more broadly protective influenza vaccine, it may be necessary to elicit antibodies that can activate effector functions in immune cells, such as antibody-dependent cellular cytotoxicity (ADCC). There is growing evidence supporting the necessity for ADCC in protection against influenza and herpes simplex virus (HSV), among other infectious diseases. An HSV-2 strain lacking the essential glycoprotein D (gD), was used to create ΔgD-2, which is a highly protective vaccine against lethal HSV-1 and HSV-2 infection in mice. It also elicits high levels of IgG2c antibodies that bind FcγRIV, a receptor that activates ADCC. To make an ADCC-eliciting influenza vaccine, we cloned the hemagglutinin (HA) gene from an H1N1 influenza A strain into the ΔgD-2 HSV vector. Vaccination with ΔgD-2::HAPR8 was protective against homologous influenza challenge and elicited an antibody response against HA that inhibits hemagglutination (HAI+), is predominantly IgG2c, strongly activates FcγRIV, and protects against influenza challenge following passive immunization of naïve mice. Prior exposure of mice to HSV-1, HSV-2, or a replication-defective HSV-2 vaccine (dl5-29) does not reduce protection against influenza by ΔgD-2::HAPR8 This vaccine also continues to elicit protection against both HSV-1 and HSV-2, including high levels of IgG2c antibodies against HSV-2. Mice lacking the interferon-α/ß receptor and mice lacking the interferon-γ receptor were also protected against influenza challenge by ΔgD-2::HAPR8 Our results suggest that ΔgD-2 can be used as a vaccine vector against other pathogens, while also eliciting protective anti-HSV immunity.

US6 Gene Deletion in Herpes Simplex Virus Type 2 Enhances Dendritic Cell Function and T Cell Activation.

Herpes simplex virus (HSV) type 1 (HSV-1) and type 2 (HSV-2) produce lifelong infections that are associated with frequent asymptomatic or clinically apparent reactivation. Importantly, HSV express multiple virulence factors that negatively modulate innate and adaptive immune components. Notably, HSV interfere with dendritic cell (DC) viability and function, likely hindering the capacity of the host to mount effective immunity against these viruses. Recently, an HSV-2 virus that was deleted in glycoprotein D was engineered (designated ΔgD-2). The virus is propagated on a complementing cell line that expresses HSV-1 gD, which permits a single round of viral replication. ΔgD-2 is safe, immunogenic, and provided complete protection against vaginal or skin challenges with HSV-1 and HSV-2 in murine models. Here, we sought to assess the interaction of ΔgD-2 with DCs and found that, in contrast to wild-type (WT) virus which induces DC apoptosis, ΔgD-2 promoted their migration and capacity to activate naïve CD8+ and CD4+ T cells in vitro and in vivo. Furthermore, DCs exposed to the WT and ΔgD-2 virus experienced different unfolded protein responses. Mice primed with DCs infected with ΔgD-2 in vitro displayed significantly reduced infection and pathology after genital challenge with virulent HSV-2 compared to non-primed mice, suggesting that DCs play a role in the immune response to the vaccine strain.

Evaluation of the Adaptive Immune Response to Respiratory Syncytial Virus.

Evaluation of the adaptive immune response is critical to the advancement of our basic knowledge and understanding of respiratory syncytial virus (RSV). The cellular composition in the lung following RSV infection is often evaluated using flow cytometry. However, a limitation of this approach has been the inability to readily distinguish cells that are within the lung parenchyma from cells that remain in the pulmonary blood vessels. Herein, we detail a procedure to evaluate the adaptive immune response via flow cytometric analysis that incorporates an in vivo intravascular staining technique. This technique allows for discrimination of immune cells in the lung tissue from cells that remain in the pulmonary vasculature following perfusion. Therefore at any given time point following an RSV infection, the leukocytic populations in the lung parenchyma can be quantified and phenotypically assessed with high resolution. While we focus on the T lymphocyte response in the lung, this technique can be readily adapted to examine various leukocytic cell types in the lung following RSV infection.

The pulmonary localization of virus-specific T lymphocytes is governed by the tissue tropism of infection.

UNLABELLED: The migration of pathogen-specific T cells into nonlymphoid tissues, such as the lung, is critical to control peripheral infections. Use of in vivo intravascular labeling of leukocytes has allowed for improved discrimination between cells located in the blood from cells present within peripheral tissues, such as the lung. This is particularly important in the lung, which is comprised of an intricate network of blood vessels that harbors a large proportion of the total blood volume at any given time. Recent work has demonstrated that >80% of antigen-specific effector CD8 T cells remain in the pulmonary vasculature following an intratracheal infection with a systemic viral pathogen. However, it remains unclear what proportion of effector CD8 T cells are located within lung tissue following a localized respiratory viral infection. We confirm that most effector and memory CD8 T cells are found in the vasculature after an intranasal infection with the systemic pathogens lymphocytic choriomeningitis virus (LCMV) or vaccinia virus (VACV). In contrast, following pulmonary viral infections with either respiratory syncytial virus (RSV) or influenza A virus (IAV), 80 to 90% of the antigen-specific effector CD8 T cells were located within lung tissue. Similarly, the majority of antigen-specific CD4 T cells were present within lung tissue during a pulmonary viral infection. Furthermore, a greater proportion of gamma interferon-positive (IFN-γ(+)) effector CD8 and CD4 T cells were located within lung tissue following a localized respiratory viral infection. Our results indicate that T cells exhibit significantly altered distribution patterns dependent upon the tissue tropism of the infection. IMPORTANCE: The migration of T cells to nonlymphoid sites, such as the lung, is critical to mediate clearance of viral infections. The highly vascularized lung holds up to 40% of blood, and thus, the T cell response may be a reflection of lymphocytes localized to the pulmonary vasculature instead of lung tissue. We examined the localization of T cell responses within the lung following either a localized or systemic viral infection. We demonstrate that following intranasal infection with a systemic pathogen, most T cells are localized to the pulmonary vasculature. In contrast, T cells are primarily localized to lung tissue following a respiratory viral infection. Our results demonstrate vast differences in the localization of T cell responses within the lung parenchyma between pathogens that can replicate locally versus systemically and that intravascular antibody labeling can be utilized to assess the localization patterns of T cell responses in nonlymphoid organs.

The CD4 T cell response to respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) can induce severe lower respiratory tract infections in infants and is the leading cause of bronchiolitis in children worldwide. RSV-induced inflammation is believed to contribute substantially to the severity of disease. T helper (Th)2-, Th9-, and Th17-related cytokines are all observed in infants hospitalized following a severe RSV infection. These cytokines cause an influx of inflammatory cells, resulting in mucus production and reduced lung function. Consistent with the data from RSV-infected infants, CD4 T cell production of Interleukin (IL)-9, IL-13, and IL-17 has all been shown to contribute to RSV-induced disease in a murine model of RSV infection. Conversely, murine studies indicate that the combined actions of regulatory factors such as CD4 regulatory T cells and IL-10 inhibit the inflammatory cytokine response and limit RSV-induced disease. In support of this, IL-10 polymorphisms are associated with susceptibility to severe disease in infants. Insufficient regulation and excess inflammation not only impact disease following primary RSV infection it can also have a major impact following vaccination. Prior immunization with a formalin-inactivated (FI-RSV) vaccine resulted in enhanced disease in infants following a natural RSV infection. A Th2 CD4 T cell response has been implicated to be a major contributor in mediating vaccine-enhanced disease. Thus, future RSV vaccines must induce a balanced CD4 T cell response in order to facilitate viral clearance while inducing proper regulation of the immune response.

Aged mice exhibit a severely diminished CD8 T cell response following respiratory syncytial virus infection.

Respiratory virus infections in the elderly result in increased rates of hospitalization and death. Respiratory syncytial virus (RSV) is a leading cause of severe virus-induced respiratory disease in individuals over the age of 65. CD8 T cells play a critical role in mediating RSV clearance. While it is clear that T cell immunity declines with age, it is not clear to what extent the CD8 T cell response to RSV is altered. Using aged BALB/c mice, we demonstrated that RSV-specific CD8 T cell responses were significantly reduced in the lungs of aged mice at the peak of the T cell response and that this decrease correlated with delayed viral clearance. Despite a decrease in the overall numbers of RSV-specific CD8 T cells during acute infection, their capacity to produce effector cytokines was not impaired. Following viral clearance, the RSV-specific memory CD8 T cells were similar in total number and phenotype in young and aged mice. Furthermore, following infection with a heterologous pathogen expressing an RSV epitope, RSV-specific memory CD8 T cells exhibited similar activation and ability to provide early control of the infection in young and aged mice. These data demonstrate a decrease in the capacity of aged mice to induce a high-magnitude acute CD8 T cell response, leading to prolonged viral replication, which may contribute to the increased disease severity of RSV infection observed for aged individuals.

Central role of dendritic cells in shaping the adaptive immune response during respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in young children. Premature infants, immunocompromised individuals and the elderly exhibit the highest risk for the development of severe RSV-induced disease. Murine studies demonstrate that CD8 T cells mediate RSV clearance from the lungs. Murine studies also indicate that the host immune response contributes to RSV-induced morbidity as T-cell depletion prevents the development of disease despite sustained viral replication. Dendritic cells (DCs) play a central role in the induction of the RSV-specific adaptive immune response. Following RSV infection, lung-resident DCs acquire viral antigens, migrate to the lung-draining lymph nodes and initiate the T-cell response. This article focuses on data generated from both in vitro DC infection studies and RSV mouse models that together have advanced our understanding of how RSV infection modulates DC function and the subsequent impact on the adaptive immune response.

Multiple CD4+ T cell subsets produce immunomodulatory IL-10 during respiratory syncytial virus infection.

The host immune response is believed to contribute to the severity of pulmonary disease induced by acute respiratory syncytial virus (RSV) infection. Because RSV-induced pulmonary disease is associated with immunopathology, we evaluated the role of IL-10 in modulating the RSV-specific immune response. We found that IL-10 protein levels in the lung were increased following acute RSV infection, with maximum production corresponding to the peak of the virus-specific T cell response. The majority of IL-10-producing cells in the lung during acute RSV infection were CD4(+) T cells. The IL-10-producing CD4(+) T cells included Foxp3(+) regulatory T cells, Foxp3(-) CD4(+) T cells that coproduce IFN-γ, and Foxp3(-) CD4(+) T cells that do not coproduce IFN-γ. RSV infection of IL-10-deficient mice resulted in more severe disease, as measured by increased weight loss and airway resistance, as compared with control mice. We also observed an increase in the magnitude of the RSV-induced CD8(+) and CD4(+) T cell response that correlated with increased disease severity in the absence of IL-10 or following IL-10R blockade. Interestingly, IL-10R blockade during acute RSV infection altered CD4(+) T cell subset distribution, resulting in a significant increase in IL-17A-producing CD4(+) T cells and a concomitant decrease in Foxp3(+) regulatory T cells. These results demonstrate that IL-10 plays a critical role in modulating the adaptive immune response to RSV by limiting T-cell-mediated pulmonary inflammation and injury.

Virally expressed interleukin-10 ameliorates acute encephalomyelitis and chronic demyelination in coronavirus-infected mice.

The absence of interleukin-10 (IL-10), a potent anti-inflammatory cytokine results in increased immune-mediated demyelination in mice infected with a neurotropic coronavirus (recombinant J2.2-V-1 [rJ2.2]). Here, we examined the therapeutic effects of increased levels of IL-10 at early times after infection by engineering a recombinant J2.2 virus to produce IL-10. We demonstrate that viral expression of IL-10, which occurs during the peak of virus replication and at the site of disease, enhanced survival and diminished morbidity in rJ2.2-infected wild-type B6 and IL-10(-/-) mice. The protective effects of increased IL-10 levels were associated with reductions in microglial activation, inflammatory cell infiltration into the brain, and proinflammatory cytokine and chemokine production. Additionally, IL-10 increased both the frequency and number of Foxp3(+) regulatory CD4 T cells in the infected central nervous system. Most strikingly, the ameliorating effects of IL-10 produced during the first 5 days after infection were long acting, resulting in decreased demyelination during the resolution phase of the infection. Collectively, these results suggest that the pathogenic processes that result in demyelination are initiated early during infection and that they can be diminished by exogenous IL-10 delivered soon after disease onset. IL-10 functions by dampening the innate or very early T cell immune response. Further, they suggest that early treatment with IL-10 may be useful adjunct therapy in some types of viral encephalitis.