Viral Immunology

Viral infections were a major cause of mortality until the use of vaccination and antiviral drugs became widespread. During the last 10 years, immunologists have made tremendous progress in dissecting mechanisms that contribute to the success of vaccines, in understanding the molecular and cellular basis of innate immunity, and in recognizing the complexity of immunological memory. Virologists have also made great progress in elucidating the molecular mechanisms of how viruses evade, manipulate, and interact with host cellular processes. In spite of these advances, most of the basic mechanisms of antiviral immunity in the host are still poorly understood due to the complexity and specificity of virus-host interactions. Here we report on advances from the recent Keystone Symposia “Viral Immunity,” organized in Keystone, Colorado on January 20–25, 2008 by Jack R. Bennink, Marcia A. Blackman, and Ann B. Hill.

Recently, it has been emphasized that chronic generalized immune activation is a leading event in the pathogenesis of HIV-1 infection. Supporting evidence comes from observations that in cases of lack of activation, infected subjects maintain a high number of T cells and do not develop AIDS-related events. Despite intensive studies, the exact mechanisms of T-cell activation are still not well understood and options for their control are limited. Very promising in this direction is a recently described T-cell subpopulation—regulatory T cells. Their functional activity and vitality are strongly dependent on the presence of IL-2. Better understanding of the mechanisms of T-cell activation, as well as the contribution of regulatory T cells to its control will increase therapeutic options for HIV-1–infected subjects. The application of immune-based therapy together with highly active antiretroviral therapy will lend a helping hand to the natural regulatory mechanisms in the control of infection.

There are few reports on the isolation, quantitative recovery, and relative purification of infectious particles that cause scrapie, Creutzfeldt-Jakob disease (CJD) and epidemic bovine spongiform encephalopathy (BSE). Because pure prion protein (PrP) has failed to show significant infectivity, it is critical to find other molecules that are integral agent components. Only complex diseased tissues such as degenerating brain have been fractionated, and agent recoveries have been quite low in concentrated abnormal prion protein (PrP-res) preparations. To simplify the purification of infectious particles, we evaluated a monotypic cell line that continuously produced high levels of the 22L scrapie agent (N2a-22L). A new rapid and accurate GT1 culture assay was used to titrate infectivity in six representative sucrose gradients. We developed a streamlined ∼3-h procedure that yielded full recovery of starting infectivity in fractions with only a few selected protein bands (representing <1% of starting protein). Infectious particles reproducibly sedimented through >30% sucrose steps, whereas PrP and PrP-res sedimentation varied depending on the conditions used. Both normal and abnormal PrP could be largely separated from infectivity in a single short centrifugation. Because no foreign enzymes were added to achieve reasonably purified infectious particles, these preparations may be used to elicit diagnostic antibodies to foreign agent proteins.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an RNA virus that initiates infection in pulmonary alveolar macrophages (PAMs), elicits weak immune responses, and establishes a persistent infection. To understand the role of dsRNA intermediates in eliciting host immunity, we sought to determine if toll-like receptor-3 (TLR3), a well-known dsRNA sensor, is involved in the regulation of PRRSV infection. TLR3 gene expression was increased in PAMs of congenitally infected 2-wk-old pigs. Stimulation of PAMs with dsRNA increased gene expression for TLR3 and interferon-β and suppressed PRRSV infectivity. To investigate activation and signaling parameters, expression constructs of wild-type and functional-domain-truncated porcine TLR3 were used in cell transfection studies. When cells that overexpressed porcine TLR3 were stimulated with dsRNA a rapid and robust calcium influx was induced. Moreover, ligand activation of porcine TLR3 expressed in MARC-145 cells elicited an antiviral response to PRRSV. Conversely, transfection of PAMs with small-interfering RNA targeting porcine TLR3 resulted in up to 80% suppression of TLR3 mRNA expression and an increase in PRRSV infectivity. These data provide fundamental genetic and molecular information for porcine TLR3, and implicate its involvement in PRRSV infection, findings that may suggest new strategies to limit this costly pandemic disease.

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be an economically important infectious disease of swine. Mechanisms governing activation of the innate immune response to PRRSV remain to be elucidated. Virulence differences observed between PRRSV isolates have been attributed to replication ability in vivo, though immunogenic differences likely contribute to virulence also. The current study utilized a single PRRSV isolate given at two different challenge doses to investigate the effect of viral replication and load on immune responses, including type I interferon activation. Body temperature, viral load, antibody levels, cellular infiltration into pulmonary tissue, and the interferon response were measured in animals receiving either a low (10² CCID₅₀) or high (10⁶ CCID₅₀) dose of inoculum to understand the role of challenge dose in acute immune responses. Initial PRRSV dose did not correlate with serum levels of PRRSV vRNA or antibody titers during the acute stage of infection (days 2–12 PI), but did have an effect on the immune response and mortality. Type I interferon responses, measured by transcriptional changes in IFN-β, IFN-α, Mx, and PKR, were uniquely different when assessed relative to viral dose or cell type, but no overall trend existed to discern responses based on challenge dose. Serum IFN-γ levels correlated with serum viral RNA load at day 19 PI. Overall, between days 2 and 12 PI, serum vRNA load was not significantly different between pigs challenged with a low or high dose of PRRSV. Animals receiving high-dose inoculum were viremic longer and eventually succumbed to respiratory disease. IFN-γ may play a role in PRRSV pathogenesis, as serum levels increased significantly in pigs challenged with the high dose of PRRSV.

We previously reported that respiratory syncytial virus (RSV) infection increases lung CD8⁺ T cell GM1 expression. The related lipid asialo-GM1 (ASGM1) is expressed by T cells in viral infection and by natural killer (NK) cells. The in vivo co-expression of GM1 and ASGM1 by immune cells is not defined. Here we analyzed lung lymphocyte GM1 and ASGM1 expression in RSV-infected mice. GM1 and ASGM1 were coordinately up-regulated by activated CD8⁺ T cells in RSV-infected BALB/c and C57BL/6 mice. In contrast, RSV infection had no effect on constitutively high NK cell GM1 expression, while increasing NK cell ASGM1 expression. GM1 and ASGM1 co-localized in lipid raft structures in NK and CD8⁺ T cells sorted from the lungs of RSV-infected mice. Anti-ASGM1 Ab treatment of RSV-infected BALB/c mice depleted GM1/ASGM1-expressing NK cells and GM1/ASGM1-expressing T cells, reduced lung IFN-γ levels, increased viral load, delayed viral clearance, and reduced illness. STAT1^–/– mice are more susceptible to RSV replication and disease than wild-type mice. In RSV-infected STAT1^–/– mice, anti-ASGM1 Ab altered cytokine levels, but in contrast to BALB/c mice, antibody treatment had no effect on viral load or illness. Taken together, GM1 and ASGM1 expression are differentially regulated by T and NK cells in RSV infection. Also, GM1/ASGM1-expressing cells are important for control of RSV in BALB/c mice, whereas STAT1^–/– mice clear RSV by an alternative pathway.

Granzyme (grz) AB^−/− H2^b mice generate numerically normal cytotoxic T lymphocyte (CTL) responses to the prominent influenza A virus D^b NP₃₆₆ and D^b PA₂₂₄ epitopes and terminate the infectious process in the pneumonic lung with the same kinetics as the WT controls. Though grz B protein expression is fully compromised, there is only a partial effect on the level of CTL activity measured in a classical, short-term ⁵¹Cr release assay. Single-cell polymerase chain reaction (PCR) analysis of both highly activated effector and “resting” memory CD8⁺ T cells from influenza A virus–infected grzAB^−/− mice showed a high prevalence of grzK mRNA⁺ expression in tetramer (tet)⁺ CTLs as was found in WT mice. However, a marked reduction in cytotoxicity present in the primary splenic CTLs of grzAB^−/− mice correlated with decreased grzK expression, as measured by real-time PCR. This suggests that grzK plays an important role in CD8⁺ T-cell cytotoxicity both in the presence and absence of grzA and B.

T-cell chemotaxis constitutes an essential function of the immune response, since active secretion of chemokines controls homing and recruitment of leukocytes into tissues. Modification of chemotactic responses by HIV-1 may provide a mechanism to increase viral spread, and may be an important factor in HIV-1 disease progression and pathogenesis. One potent T-cell chemoattractant is SDF-1α, the natural ligand for the HIV-1 co-receptor CXCR4. In addition, the HIV-1 gp120 molecule shares the chemotactic properties of several chemokines, including SDF-1α. HIV-1 Nef is a pathogenic determinant and a virulence factor that has pleiotropic effects on immune cell processes and receptor signaling. In this study, the effects of Nef on T-cell migration to SDF-1α, and on CXCR4 receptor signaling were examined. We report that disruption of the proline-rich region of Nef inhibits T-cell migration to SDF-1α. This dominant negative effect indicates that Nef occupies a position in the CXCR4-mediated signaling pathway that is upstream of an SH3-dependent pathway. The results suggest that Nef may play an important role in homing of T cells during viral invasion in HIV-1 disease.

Marburg virus (MARV) causes a severe and usually lethal hemorrhagic disease in humans and non-human primates. Here, 16 cynomolgus macaques were experimentally infected with the Ci67 strain of MARV. Blood and spleen samples were collected at various time points after infection to study the immunological response to MARV. Beginning at day 2 and continuing throughout the course of the infection there was a rise in antigen-presenting cells in both the blood and spleen expressing MARV glycoprotein. Natural killer (NK) cells declined in the blood after infection (from 15% on day 0 to 5% on day 6), but a small increase was seen in the spleen samples. Little or no change in CD4⁺ or CD8⁺ T cells was observed out to day 6 post-exposure in blood, while there was a continual decline in the percentage of CD8⁺ T cells in spleen samples. Circulating B cells (defined as CD20⁺) increased during the course of the infection as did CD4⁺ CD8⁺ (double-positive) T cells. Intracellular cytokine staining indicated that by day 6 a large population of leukocytes in the spleen were producing IFN-α; analysis of surface markers indicated that these cells were plasmacytoid dendritic cells based on their expression of CD123⁺, but these cells had decreased expression of class II MHC. IL-6 production was detected late in the infection in CD14⁺ spleen cells. These results suggest a robust innate immune response to MARV; however, this response was delayed relative to the infection.

The determination of immunogenic peptides of hepatitis C virus (HCV) is pivotal for vaccine development. We previously reported that the majority of patients infected with HCV have significant levels of IgG specific to an HCV-derived peptide at positions 35–44 of core protein (C35–44), a major epitope recognized by cellular immunity. This study addresses whether or not the other subclasses of immunoglobulins to this peptide exist. As a result, IgE, but not IgM or IgA, specific to this peptide is consistently detectable in the majority of patients with HCV infection, regardless of the different HLA types and disease conditions. These results provide additional information on this immunogenic peptide with new insights that contribute to a better understanding of host responses to HCV.

Persistent cervicovaginal infection with high-risk types of HPV is the major risk factor for subsequent cervical neoplasia. HPV53, part of the α6 species group along with HPV types 30, 56, and 66, is one of the most prevalent high risk–related HPV types, yet little is known about the molecular basis of its benign behavior. We generated and utilized HPV53 virus-like particles (VLPs) to investigate risk factors for its seroprevalence in a population of young college women. Seropositivity to HPV53 VLPs was determined using a polymer-based ELISA to measure IgG reactive antibodies. Cervicovaginal cells were collected for HPV DNA detection and typing by MY09/11 PCR. A questionnaire queried for HPV risk factors to estimate odds ratios (ORs). Prevalence of cervicovaginal HPV DNA was 26% (n = 148); 3% of women (n = 17) had HPV53 DNA and 7% (n = 40) were seropositive to HPV53. Seroprevalence of IgG to HPV53 VLPs in women with cervicovaginal HPV53, HPV53-related types (HPV30, 55, and 66), other HPV types, and no HPV was 41%, 11%, 7%, and 6%, respectively (p _trend < 0.001). Risk factors independently associated with HPV53 VLP seropositivity included use of oral contraceptive pills (OCPs) (OR: 4; 95% CI: 1.8, 9), having ≥2 regular partners in the last 6 months (OR: 2.5; 95 % CI: 1.1, 5.8), having a regular male partner with ≥4 lifetime sex partners (OR: 2.6; 95% CI: 1.1 6), seropositivity to HPV16 (OR: 6.7; 95% CI: 3.1, 14.5), and isolation of HPV53 DNA from cervicovaginal lavage (OR: 17.3; 95% CI: 5.3, 55.9). In conclusion, host serological responses to HPV53 VLPs are strongly type-specific, and subjects' risk for HPV53 seropositivity is independently associated with sexual behavior and OCP use.

Hashimoto's thyroiditis is a common autoimmune disorder of the thyroid gland. It has been linked to infections with hepatitis C, EBV, HTLV-1, and Yersinia enterocolitica. As parvovirus B19 has been associated with a wide spectrum of autoimmune diseases, we investigated the potential role of B19 infection in inducing Hashimoto's thyroiditis. Serum samples derived from 73 children and adolescents with Hashimoto's thyroiditis and from 73 age-matched controls were included in the study. The mean age of disease manifestation was 10 y 7 mo. All samples were analyzed for the presence of viral DNA and for antibodies against VP1, VP2, and NS1 proteins. VP1- and VP2-specific antibodies were present in 38 patients (52%) and 43 controls (59%; N.S.). NS1-specific antibodies were detectable in 23 patients (32%) and 19 controls (26%; N.S.). Parvovirus B19 DNA was detectable in 9 patients (12%) and 2 controls (3%; p < 0.03), indicating recent B19-infection. A negative correlation between disease duration and the detection of viral DNA was seen. The mean disease duration in B19-DNA-positive patients was 6 mo, compared to 29 mo in the remainder (p < 0.01).

There is strong evidence that acute parvovirus B19 infections are involved in the pathogenesis of some cases of Hashimoto's thyroiditis.

IL-21 is a relatively newly discovered multifunctional and pleiotropic cytokine. It is produced primarily by CD4⁺ T cells, the principal targets of the virus, and therefore this cytokine has special relevance to HIV infection. Here we show for the first time that serum levels of this cytokine are significantly reduced in HIV-infected AIDS patients and correlate significantly with their CD4⁺ T-cell counts. These data suggest that the cytokine levels could act as a valuable biomarker for the progression of AIDS.