Viral Immunology

Hepatitis C virus (HCV) infection in humans is almost invariably associated with viral persistence and chronic hepatitis. HCV-induced chronic hepatitis is a major risk factor for the development of hepatocellular carcinoma. The high incidence of HCV persistence suggests that this virus has evolved one or more mechanisms to evade and possibly suppress host immune responses. To understand the mechanism(s) involved in the establishment of HCV persistence, we have identified an HCV core protein as an immunomodulatory molecule to suppress host immune response. We have further determined a molecular mechanism of HCV core-mediated immune suppression by searching for a potential host protein(s) capable of associating with the HCV core protein. Interestingly, the C1q complement receptor, gC1qR, can bind to the HCV core. C1q is a ligand of gC1qR and is involved in the early defense against viral infection as well as regulation of adaptive immune response. Similar to C1q, the HCV core can inhibit human T-lymphocyte proliferative response through its interaction with the gC1qR. It implicates that HCV core/gC1qR-induced immune suppression may play a critical role in the establishment of persistent infection.

Most strategies for reducing global measles morbidity and mortality and eliminating measles are based on the ability to enhance immune responses to measles virus. Challenges to measles elimination and eradication are based in part on the need to sustain high levels of population immunity to interrupt transmission of measles virus. We review aspects of the immunology of measles and measles vaccination with the aim of demonstrating how knowledge of the immune responses is essential to furthering the goals of reducing measles morbidity and mortality and the elimination of measles. Better understanding of the mechanisms of immune suppression after measles, the potential for alternative vaccination strategies to induce immunity in young infants, and the immunologic basis of atypical measles, increased mortality after high-titer measles vaccine, and waning immunity will lead to improved strategies for measles control and elimination.

West Nile virus (WNV) first appeared in the naive environment of the Western Hemisphere in 1999 in New York. Genetic analysis determined that the virus was introduced into the United States from the Mediterranean Basin. This review discusses the spread of the virus in 2001 from the initial focus in Queens, New York, to widespread activity in the eastern and midwestern United States. It concentrates on viral ecology, epizootiology, pathology, prediction, and prevention. Research questions to further our understanding of the transmission cycle of WNV are discussed, including host-preference studies, molecular confirmation of implicated mosquito vectors, and survival of WNV in the temperate environment of the United States. Comparisons are drawn with two other arboviruses enzootic in the United States, eastern equine encephalitis, and St. Louis encephalitis viruses. Although not recently introduced, these two viruses also demonstrated increased activity in the United States in 2001.

A versatile DNA vaccine (pdIV3) was constructed by replacing the integrase, vif, vpx, and vpr genes of a pathogenic simian immunodeficiency virus (SIV) molecular clone with a linker containing unique cloning sites. The 5′ long terminal repeat (LTR) is truncated and transcription is controlled by a cytomegalovirus (CMV) promoter. The construct expresses Gag and Env in vitro and noninfectious virus particles are produced from transfected cells. The ability of pdIV3 to promote cellular and humoral immune responses, along with the flexibility of the linker design to allow insertion of immunostimulatory genes in future constructs, makes this a useful base vector for immunization against primate lentiviruses. We present the construction of a retroviral plasmid designed to serve as a template for the development of safe and effective vaccines against primate immunodeficiency retroviruses. This vaccine component should facilitate the simultaneous induction of cellular and humoral immune responses that protect primates against infection with SIV and human immunodeficiency virus (HIV) and the development of acquired immune deficiency syndrome (AIDS). This plasmid could induce the appropriate immune response required to attack both cell-free and cell-associated viruses. The lack of infectivity, the inability to integrate, and the SIV origin make this construct a safe alternative to attenuated vaccines based on HIV. In addition, we intend to develop this construct as an immunotherapeutic approach to lower the viremia in AIDS patients.

Rinderpest virus (RPV), a member of the genus Morbillivirus within the Paramyxoviridae family, causes a highly contagious and often fatal disease known as rinderpest in wild and domestic ruminants. The envelope of the virus contains two surface glycoproteins, namely the hemagglutinin (H) and the fusion (F) proteins, both of which have been shown to confer protective immunity in animals. In this paper, we demonstrate that single administration of low doses of recombinant H protein of RPV expressed in insect cells in the form of extracellular virus induces long lasting bovine leukocyte antigen class I restricted cytotoxic T-cell (CTL) responses in cattle in the absence of adjuvant. This is the first report of CTL responses in cattle against one of the protective antigens of RPV.

DNA immunization was used to compare the immunogenicity of hepatitis B virus S gene variants. Four recombinant plasmid DNAs containing the full-length virus genome with different S gene inserts were used to immunize BALB/c and C57/BL/6 mice. These inserts were cloned from 129L (residue 129, glutamine to leucine), 129H (residue 129, glutamine to histidine) 145R (residue 145, glycine to arginine) variants and the wild-type virus. The titer of hepatitis B virus core antibodies (anti-HBc) in immunized mice was used as the control for the efficiency of DNA immunization. Serum hepatitis B surface antibody (anti-HBs) titer and cytokines induced in splenocytes stimulated with hepatitis B surface antigen (HBsAg) were monitored as specific immune responses induced by different plasmid DNAs. 129L DNA induced significantly lower anti-HBs antibodies (IgG, IgG1, and IgG2a) and less interferon-γ, compared to those in mice immunized with the 129H variant and the wild-type HBV DNA (p < 0.05). Computer modeling showed that a change from glutamine to leucine at 129 residue led to higher hydrophobicity and could result in decreased immunogenicity. Results indicate that DNA immunization can be used to compare the humoral and cellular immunogenicity among different HBV S variants.

Among cytomegalovirus (CMV) tegument proteins, phosphoprotein 65 (pp65) has been identified as the important target antigen of the cytotoxic T lymphocyte (CTL) response against the virus. We synthesized seven CMV-pp65-derived peptides carrying an HLA-A24-binding motif, and investigated the ability of these peptides to induce CMV-specific CTL. We identified one nonamer peptide (pp65_113-121; VYALPLKML) able to bind HLA-A24 and induce CTL responses in vitro in peripheral blood mononuclear cells (PBMC) from CMV-seropositive individuals. The peptide-specific CTLs generated were capable of recognizing pp65 expressed on CMV-infected fibroblasts as well as pp65_113-121 peptide bound to the surface of C1R-A*2402 cells in an HLA-A24-restricted manner. The pp65_113-121 peptide thus might be considered a synthetic peptide vaccine in HLA-A24–positive individuals.

Integration of human immunodeficiency virus type-1 (HIV-1) proviral DNA into host cell genomic DNA ensures viral persistence despite suppression of active replication. Because HIV RNA originates from integrated HIV DNA, HIV RNA and DNA loads should interrelate when suppression of viral replication is incomplete. In addition, the link between proviral DNA formation and generation of HIV-1 genetic diversity suggests that the ease with which HIV escapes immune or drug-based suppression should vary with proviral load. Thus, HIV proviral load should have unique prognostic significance independent of the highly labile plasma HIV RNA levels commonly used to monitor patient status. To test this possibility, we developed a simple standardized research assay estimating the proportion of CD4⁺ peripheral blood mononuclear cells (PBMC) carrying HIV-1 DNA and investigated associations between this parameter, plasma virus load, long-term efficacy of antiretroviral therapy and restoration of CD4⁺ T cells. Lower proportions of CD4⁺ PBMC carrying HIV-1 DNA were associated with lower peak plasma HIV RNA levels and with more favorable long-term responses to antiretroviral therapy. These results suggest that HIV proviral load affects both disease progression and responsiveness to antiretroviral therapy. Therefore, new anti-HIV therapies addressing the stable pool of HIV proviral DNA should be developed to improve long-term prospects for suppression of HIV replication.

Intranasal infection of mice with murine γ-herpesvirus 68 (MHV-68) elicits a striking CD8⁺ T-cell lymphocytosis following the establishment of latency, which includes a marked increased frequency of Vβ4⁺ CD8⁺ T cells. The Vβ4⁺ CD8⁺ T cells do not recognize a conventional viral peptide, but are stimulated by an uncharacterized ligand expressed on latently infected, activated B cells. The selective expansion of Vβ4⁺ CD8⁺ T cells after MHV-68 infection is observed in all mouse strains examined, although the fold-increase varies widely, ranging from less than twofold to greater than 10-fold. The factors controlling the variation are currently undefined. In the current study, CD8⁺ T cell activation and Vβ4⁺ CD8⁺ T-cell frequencies were analyzed in 18 inbred strains of mice. The data show that the magnitude of the Vβ4⁺ CD8⁺ T-cell response correlates with the degree of CD8⁺ T cell-activation, and that both major histocompatibility complex (MHC) and non-MHC genes contribute to the magnitude of the activation. Furthermore, the magnitude of the response does not reflect major differences in susceptibility to viral infection and/or corresponding differences in the acute response. Rather the degree of Vβ4⁺ CD8⁺ T cell activation may be determined by differences in levels of expression of the stimulatory ligand at the peak of latency.

Although nearly all adults are seropositive for adenoviruses, little is known about the cellular immune responses to these ubiquitous pathogens. We have previously identified adenovirus-specific proliferative T-cell responses in peripheral blood mononuclear cells (PBMC) from healthy adults. In this study, memory T-cell responses to adenovirus were further evaluated in healthy adult donors using a short term, quantitative enzyme-linked immunospot assay (ELISPOT) assay. Adenovirus antigen induced specific secretion of interferon-γ (IFN-γ) from PBMC within 12 hours of incubation. PBMC from 20 of 22 healthy donors (90.9%) expressed IFN-γ in response to adenovirus. Responder cells were identified as CD4⁺ T cells by immunomagnetic depletion methods. Interleukin-4(IL-4) secretion was not detected, consistent with a TH1 response. There was a 10-fold variation in the frequencies of adenovirus-specific CD4⁺ T cells between donors (range, 34 to 294; median, 122 per million PBMC). Adenovirus-specific T cell frequencies remained stable over periods up to 2 years among individual donors, but there was an inverse correlation between frequency and donor age. These quantitative data suggest that most adults retain adenovirus-specific cellular memory after childhood exposure. This assay may be useful for the evaluation of adenovirus-specific CD4⁺ T-cell responses in patients treated with adenovirus gene therapy vectors and the identification of major T-cell epitopes.

Serum antibodies against the E6 and E7 proteins of human papillomavirus (HPV) 16 and 18 are associated with cervical cancer. The aim of this study was to investigate the presence of local antibodies against HPV in cervicovaginal washings (CWs). In this study antibodies against the native HPV16 and HPV18 E6/E7 proteins were detectable in CWs (48%) and sera (29%) from patients with cervical cancer (n= 21) utilizing a sandwich protein enzyme-linked immunosorbent assay (ELISA). In paired CWs and sera from patients with cervical intraepithelial neoplasia (n = 38) and from healthy women (n= 22) no antibodies against these proteins were found. In 10 of 11 patients, the antibody response corresponded with the HPV type in the cervical smear and/or tumor tissue, which indicates the HPV type specificity of the assay. In 7 of 11 patients with antibody reactivity against HPV16 or HPV18 E6 and/or E7 proteins a higher level of antibody reactivity in the CWs than in the paired serum samples was found at similar inputs of total IgG. This suggests that the antibodies in the CWs against the investigated HPV proteins in these patients were locally produced.