Viral Immunology

Objective: The aim of this present study was to use Luminex technology to detect antibodies against the late antigen L1 as well as those directed against the early antigens E6 and E7. Background Data: Human papillomavirus (HPV) serology is complex because infection and disease lead to distinct type-specific antibody responses. Materials and Methods: Viral antigens were expressed with pGEX vectors in Escherichia coli and then used in Luminex as coating antigens for antibody detection in 205 human sera samples: 71 cervical cancer cases, 64 cases of cervical inflammation, and 70 controls. Results: The data showed that 90.14% of sera among the cervical cancer patients had seropositivity toward at least one of the HPV 16 or HPV 18 antigens. Moreover, the percentages of positivity toward E6 and E7 HPV 16 antigens were 44% and 61%, respectively, versus only 21% for the L1 antigen. Among cervical cancer patients, the data showed different distributions in women of different ages. In addition, the intensity of the antibody response was also different for the six antigens analyzed. Conclusions: Antibody detection depends on the type of antigen, and is well correlated with international scientific findings. The differences in antibody response between patients with inflammation and patients with cervical cancer were significant.

The epidemiology of West Nile virus (WNV) in Ghana, sub-Saharan Africa, and its relevance to transfusion were newly assessed. A total of 1324 plasma samples from five Ghanaian populations, including 529 children (<6 y old, pre-transfusion) and 795 adults (236 blood donors, 226 HIV-infected or non-infected pregnant women, 203 HIV symptomatic patients, and 130 AIDS patients) were screened for WNV RNA. No WNV RNA was detected, but 4.8% (13/271) and 27.9% (127/455) carried specific IgG in children and adults, respectively, and 2.4% (4/167) of the children had IgM. The prevalence of IgG antibody to WNV increased progressively and peaked around 30% between ages 1 and 30 y, then stabilized. The absence of viremia in four WNV IgM-positive children, and of reactivation in HIV-infected patients suggests that once host immunity is established, it appears to be robust. In addition, there were no clinical reports of WNV infection in the hospital in Kumasi, Ghana, suggesting that WNV epidemiology in Ghana differs from that seen in the U.S. Most infections occur early in life, and as the window for infection is quite short, the risk of transmission by transfusion appears to be low, and the risk of pathogenicity in immunocompetent recipients appears to be limited in an endemic area such as Ghana.

For several years, researchers have known that the generation of neutralizing antibodies is a prerequisite for attaining adequate protection against dengue virus. Nevertheless, the cellular immune response is the principal arm of the adaptive immune system against non-cytopathic viruses such as dengue, as once the virus enters into the cell it is necessary to destroy it to eliminate the virus. To define the role of the cellular immune response in the protection against dengue, we selected the mouse encephalitis model. Mice were immunized with a single dose of infective dengue 2 virus and different markers of both branches of the induced adaptive immunity were measured. Animals elicited a broad antibody response against the four dengue virus serotypes, but neutralizing activity was only detected against the homologous serotype. On the other hand, the splenocytes of the infected animals strongly proliferated after in vitro stimulation with the homologous virus, and specifically the CD8 T-cell subset was responsible for the secretion of the cytokine IFN-γ. Finally, to define the role of T cells in in vivo protection, groups of animals were inoculated with the depleting monoclonal antibodies anti-CD4 or anti-CD8. Only depletion with anti-CD8 decreased to 50% the level of protection reached in the non-depleted mice. The present work constitutes the first report defining the role of the cellular immune response in protection against dengue virus in the mouse model.

Specific-pathogen free chickens were infected with the RB1B strain of Marek's disease virus (MDV) and T cells from the spleens of infected as well as age-matched controls were fractionated by flow cytometry at 4, 10, and 21 days post-infection (d.p.i.). Real-time quantitative reverse transcription PCR was used to assess the amount of cytokine transcripts as well as viral genes meq and glycoprotein B (gB). There was an increase in the number of CD4⁺ T cells, as well as a significant increase in the expression of the viral meq gene in CD4⁺ T cells, which coincided with the presence of tumors in various organs of infected birds. It was also observed that there was a significant upregulation in the amount of the gene expression of interferon (IFN)-γ, interleukin (IL)-18, and IL-6 at 4 and 21 d.p.i. in CD4⁺ and CD8⁺ T-cell subsets. The expression of IL-10 was upregulated as well. The outcome of the cytokine milieu inclined towards the induction of a type I immune response at 4 and 21 d.p.i. Our study indicates that MDV-associated cytokine profiles vary in CD4⁺ and CD8⁺ T-cell subsets, and that cytokines including IFN-γ, IL-18, IL-6, and IL-10 may play a role in the elicitation of an immune response to MDV.

While hepatitis C virus (HCV)-specific immune responses are attenuated in HCV/HIV co-infected patients compared to those infected with HCV alone, the reasons for this remain unclear. In this study, the proportions of regulatory, naïve, and memory T cells, along with chemokine receptor expression, were measured in co-infected and mono-infected patients to determine if there is an alteration in the phenotypic profile of lymphocytes in these patients. HCV/HIV co-infected patients had increased proportions of CD4⁺ naïve cells and decreased proportions of CD4⁺ effector cells when compared to HCV mono-infected patients. The proportions of CD4⁺ Tregs and CD4⁺ CXCR3⁺ T cells were also significantly lower in co-infected patients. A decrease in CD4⁺ Tregs and subsequent loss of immunosuppressive function may contribute to the accelerated progression to liver disease in co-infected individuals. Dysregulation of immune responses following reduction in the proportions of CD4⁺ CXCR3⁺ Th-1 cells may contribute to the reduced functional capacity of HCV-specific immune responses in co-infected patients. The findings of this study provide new information on the T-cell immunophenotype in HCV/HIV co-infected patients when compared to those infected with HCV alone, and may provide insight into why cell-mediated immune responses are diminished during HCV infection.

Virus-like particles (VLPs) are highly immunogenic. In this study, gene fragments encoding hepatitis A virus (HAV) vp1 (aa 24–171), a hepatitis E virus (HEV) ORF2 gene fragment encoding aa 431–615, and gene fragments encoding a nine-peptide linker were spliced together. The spliced gene fragments were then inserted into the expression vector pBV220, resulting in the recombinant plasmid pBV-EA342, which expressed a 342-amino acid fusion protein. The fusion protein was expressed in Escherichia coli and specifically reacted to human hepatitis A- and E-positive sera. VLPs were formed during the renaturation of the EA342 fusion protein. The chimeric HAV and HEV VLPs were able to immunize KM mice, and they induced strong anti-HAV and anti-HEV humoral immune responses. These results are promising for future studies.

The spike (S) glycoprotein of severe acute respiratory syndrome coronavirus (SARS-CoV) is a major target in the development of diagnostic assays and vaccines, but its antigenic and immunogenic properties remain unclear. Seven SARS-CoV spike proteins (S, SQ, S1, RBD, S2, S2Q, and CX) were generated using the modified vaccinia virus (Tiantan strain) as a vector, and their antigenicity and immunogenicity were evaluated. The secreted SQ protein in which the transmembrane domain was deleted, as well as the full-length spike protein, showed the most potential to induce the production of neutralizing antibody (nAb) in mice. S1 and RBD proteins initialized significantly lower levels of nAb production. In addition, the S proteins were recognized specifically by the sera of convalescent patients with SARS, and that of mice immunized with inactivated SARS-CoV, but did not react with anti-sera of HCoV-OC43 or HCoV-229E, or sera from healthy donors (although RBD showed a false-positive in 1 of 55 control samples of human sera). Our results demonstrate that SQ protein may be an effective vaccine candidate and a convenient and safe diagnostic antigen for SARS-CoV.

Herpesviruses are widely disseminated in the population and establish lifelong latency, which is associated with a variety of pathological consequences. A recent report showed that mice latently infected with either murine γ-herpesvirus-68 (γHV68) or murine cytomegalovirus (mCMV), mouse pathogens genetically similar to the human herpesviruses, Epstein-Barr virus, Kaposi's sarcoma–associated herpesvirus, and cytomegalovirus, had enhanced resistance to subsequent bacterial infection, suggesting protective as well as deleterious effects of latency. Here we confirm that latent γHV68 infection confers protection against subsequent infection with Listeria monocytogenes. However, the effect is transient, lasting only a few months.