Frances Gotch

Cytotoxic T-Cell Immunity to Influenza

In a study designed to determine whether cytotoxic T lymphocytes contribute to immunity against influenza virus infection, we inoculated 63 volunteers intranasally with live unattenuated influenza A/Munich/1/79 virus. Over the next seven days clinical observations were made, and the amount of virus shed was measured. The protective effects of preinfection serum antibody and of cytotoxic T-cell immunity against influenza A virus were assessed for each participant. All subjects with demonstrable T-cell responses cleared virus effectively. This response was observed in volunteers in all age groups, including those born after 1956, who did not have specific antibody and hence had probably not been exposed to this subtype of influenza A virus before. Cytotoxic T cells show cross-reactivity in their recognition of the different subtypes of influenza A virus, in contrast to the antibody response that is specific for each virus subtype. We conclude that cytotoxic T cells play a part in recovery from influenza virus infection.

miR-132 regulates antiviral innate immunity through suppression of the p300 transcriptional co-activator

MicroRNAs are small, non-coding RNAs that negatively regulate gene expression. It has been proposed that microRNAs could function in the regulation of innate immunity, but this has not been demonstrated for viral infection. Here we test this hypothesis using the human pathogenic virus Kaposis sarcoma-associated herpesvirus (KSHV) and one of its putative natural cellular targets, primary lymphatic endothelial cells (LECs). We show that an early antiviral microRNA response (6 h post-infection) includes expression of microRNAs that enhance viral gene expression. In particular, the CREB-induced miR-132 microRNA is highly upregulated after infection and has a negative effect on the expression of interferon-stimulated genes, facilitating viral replication. We show a similar function for miR-132 during infection of monocytes with herpes simplex virus-1 (HSV-1) and human cytomegalovirus (HCMV). miR-132 regulates innate antiviral immunity by inhibiting expression of the p300 transcriptional co-activator. p300 is downregulated early after KSHV infection, and inhibition of miR-132 induction restores p300 expression. Furthermore, p300 regulates miR-132 levels, revealing a dynamic equilibrium between miR-132 and p300. By targeting p300, rather than a transcription factor or signalling protein, miR-132 has a broad role in the regulation of antiviral immunity.

Plasmacytoid Dendritic Cells Are Highly Susceptible to Human Immunodeficiency Virus Type 1 Infection and Release Infectious Virus

ABSTRACT Plasmacytoid dendritic cells (pcDC) and myeloid dendritic cells (myDC) are shown to express CD4 and low levels of CCR5 and CXCR4, but only myDC express DC SIGN, a C-type lectin that binds human immunodeficiency virus but does not mediate virus entry. Both DC types were more susceptible to infection with a macrophage than a lymphotropic strain of human immunodeficiency virus type 1, but pcDC were more readily infected than myDC.

Loss of Discrete Memory B Cell Subsets Is Associated with Impaired Immunization Responses in HIV-1 Infection and May Be a Risk Factor for Invasive Pneumococcal Disease

Invasive pneumococcal infection is an important cause of morbidity and mortality in HIV-1-infected individuals. B cells play an important role in maintaining serologic memory after infection. IgM memory B cells are significantly reduced in HIV-1-infected patients and their frequency is similar to that observed in other patient groups (splenectomized individuals and patients with primary Ab deficiency) who are also known to have an increased risk of invasive pneumococcal infection. Antiretroviral therapy does not restore marginal zone B cell percentages. Immunization with the 23-valent polysaccharide pneumococcal vaccine shows that HIV-1-infected patients have impaired total IgM and IgG pneumococcal vaccines compared with healthy controls. Loss of switched memory B cells was associated with impaired tetanus toxoid IgG vaccine responses. Results of this study demonstrate that defects in B cell memory subsets are associated with impaired humoral immune responses in HIV-1 patients who are receiving antiretroviral therapy and may be a contributory factor to the increased risk of invasive pneumococcal infection observed in HIV-1 infection.

Loss of CD4+ T Cell Proliferative Ability but Not Loss of Human Immunodeficiency Virus Type 1 Specificity Equates with Progression to Disease

In this study, we compared human immunodeficiency virus (HIV) type 1-specific proliferative responses with HIV-1-induced intracellular cytokine production in a cohort of clinically nonprogressing patients and individuals with progressive HIV-1 infection. We found strong HIV-1-specific proliferative responses in the clinical nonprogressor cohort that correlated with significant numbers of circulating HIV-1-specific CD4(+) T cells. In contrast, HIV-1-specific proliferative responses were absent in most individuals with progressive HIV-1 infection, even though interferon-gamma-producing HIV-1-specific CD4(+) T cells were detectable by flow cytometry. The implication of these data is that the important dysfunction seen in most HIV-positive patients from very early in disease may be an inability of HIV-1-specific CD4(+) memory T cells to proliferate in response to HIV antigens rather than an absolute loss of circulating virus-specific CD4(+) T cells.

Adenovirus vector vaccination induces expansion of memory CD4 T cells with a mucosal homing phenotype that are readily susceptible to HIV-1

In the recently halted HIV type 1 (HIV-1) vaccine STEP trial, individuals that were seropositive for adenovirus serotype 5 (Ad5) showed increased rates of HIV-1 infection on vaccination with an Ad5 vaccine. We propose that this was due to activation and expansion of Ad5-specific mucosal-homing memory CD4 T cells. To test this hypothesis, Ad5 and Ad11 antibody titers were measured in 20 healthy volunteers. Dendritic cells (DCs) from these individuals were pulsed with replication defective Ad5 or Ad11 and co-cultured with autologous lymphocytes. Cytokine profiles, proliferative capacity, mucosal migration potential, and susceptibility to HIV infection of the adenovirus-stimulated memory CD4 T cells were measured. Stimulation of T cells from healthy Ad5-seropositive but Ad11-seronegative individuals with Ad5, or serologically distinct Ad11 vectors induced preferential expansion of adenovirus memory CD4 T cells expressing α4β7 integrins and CCR9, indicating a mucosal-homing phenotype. CD4 T-cell proliferation and IFN-γ production in response to Ad stimulation correlated with Ad5 antibody titers. However, Ad5 serostatus did not correlate with total cytokine production upon challenge with Ad5 or Ad11. Expanded Ad5 and Ad11 memory CD4 T cells showed an increase in CCR5 expression and higher susceptibility to infection by R5 tropic HIV-1. This suggests that adenoviral-based vaccination against HIV-1 in individuals with preexisting immunity against Ad5 results in preferential expansion of HIV-susceptible activated CD4 T cells that home to mucosal tissues, increases the number of virus targets, and leads to a higher susceptibility to HIV acquisition.

Prospective study of the effects of antiretroviral therapy on Kaposi sarcoma--associated herpesvirus infection in patients with and without Kaposi sarcoma.

Objective: To investigate the effect of highly active antiretroviral therapy (HAART) on circulating Kaposi sarcoma (KS)‐associated herpesvirus (KSHV) load in HIV‐infected individuals with and without KS. Design: Twenty‐nine HIV‐infected subjects (21 with KS and 8 without KS) were recruited for the study; they were prospectively studied before and at regular intervals during HAART. Methods: Patients with KS were clinically assessed using Adult Clinical Trials Group (ACTG) criteria, and sequential blood samples were obtained from all patients for determination of plasma HIV‐1 load, CD4 cell count, titer of antibody to KSHV, and KSHV load. Results: Ten of 21 patients with KS had a favorable KS response (complete response, 6; partial response, 4) with HAART alone. Of the 20 subjects with detectable KSHV viremia prior to HAART, over one half (12 [60%]) had an undetectable KSHV load with antiretroviral therapy. There was no significant difference between subjects receiving protease inhibitor‐ or nonnucleoside reverse transcriptase inhibitor‐based treatment combinations. Achieving undetectable KSHV viremia is associated with a better clinical outcome in patients with KS. Conclusions: To our knowledge, we demonstrate for the first time that both protease inhibitor‐based and nonnucleoside reverse transcriptase inhibitor‐based antiretroviral treatment combinations may lead to an undetectable KSHV load and confirm that an undetectable KSHV load is associated with KS regression. There was no clear association between CD4 cell count response and KS response to HAART, but there was a significant relationship between HIV load response to HAART and clinical improvement of KS.

Identification of Kaposi's Sarcoma-Associated Herpesvirus (KSHV)-Specific Cytotoxic T-Lymphocyte Epitopes and Evaluation of Reconstitution of KSHV-Specific Responses in Human Immunodeficiency Virus Type 1-Infected Patients Receiving Highly Active Antiretroviral Therapy

ABSTRACT Following the introduction of highly active antiretroviral therapy (HAART), the incidence of Kaposis sarcoma (KS) has significantly declined in human immunodeficiency virus type 1 (HIV-1)-positive (HIV-1+) individuals and clinical remission is often observed. We hypothesize that these effects are partly due to anti-KS-associated herpesvirus (KSHV) immune restoration. Here, 15-mer overlapping peptides from proteins K12 and K8.1 were used to identify novel KSHV-specific cytotoxic T-lymphocyte epitopes. Three immunogenic peptides, two lytic and one latent, were subsequently used to monitor the anti-KSHV CD8+ T-cell responses in a cohort of 19 HIV-1+ KSHV+/− KS+/− individuals during 52 weeks of HAART. KSHV and HIV-1 loads, KSHV antibody titers, and both CD4+ and CD8+ T-lymphocyte counts were enumerated. Prior to HAART, the total number of spot-forming cells (SFC) for all three peptides correlated with both CD4+ and CD8+ T-lymphocyte counts (P ≤ 0.05) in the KSHV-positive KS-positive cohort (n = 11). Following 52 weeks of HAART, significant decreases in HIV-1 and KSHV loads were associated with significant increases in CD4+ T-lymphocyte counts and number of SFC for the three KSHV-specific peptides. Although these increases were modest in comparison to the number of SFC observed with the HIV-1 gag peptide SLYNTVATL, they represented a fourfold increase from the baseline, continuing an upward trend to week 52.

A Balanced Type 1/Type 2 Response Is Associated with Long-Term Nonprogressive Human Immunodeficiency Virus Type 1 Infection

ABSTRACT Previous reports have emphasized the requirements for strong type 1 cell-mediated responses in the control of human immunodeficiency virus type 1 (HIV-1). HIV-1 Gag p24-specific CD4 helper T-lymphocyte (HTL) responses have been shown to inversely correlate with viral burden in HIV-1-infected individuals. In this study, peripheral blood mononuclear cells from 70 individuals with chronic progressive HIV-1 infection (clinical progressors), 10 clinical nonprogressors, and 3 immunologically discordant progressors were assessed for HTL proliferation and type 1/type 2 cytokine production. Clinical progressors lacked functional HIV-1-specific HTLs with proliferative and cytokine-producing capacity. Clinical nonprogressors were found to respond to a wide range of HIV-1 antigens from different clades, producing both type 1 and type 2 cytokines. Immunologically discordant progressors responded strongly to clade B Gag p24 with a type 1 cytokine profile but not to other antigens. Thus, in contrast to clinical nonprogressors, neither progressors nor immunologically discordant progressors secreted interleukin-4 (IL-4) in response to HIV-1 antigens. Both clinical nonprogressors and immunologically discordant progressors responded broadly to B clade Gag p24-overlapping peptides. However, IL-4 production in the nonprogressors was restricted to a limited number of p24 peptides. No HIV-1-specific T-cell responses were seen in 20 seronegative controls. Additionally, we observed a rapid type 1 to type 2 shift in the response of one immunologically discordant progressor upon onset of clinical symptoms. These results suggest that a balanced type 1/type 2 profile correlates with successful long-term control of HIV-1.

Assessment of type 1 and type 2 cytokines in HIV type 1-infected individuals : Impact of highly active antiretroviral therapy

Although the effect of highly active antiretroviral therapy (HAART) on HIV-1 replication has been established, the mechanisms involved in restoration of immune responses and reconstitution remain unknown. This study provides evidence of changes in expression of type 1 and type 2 cytokine-specific mRNA occurring during HIV-1 infection, before and after initiation of HAART. Unstimulated PBMCs from nine HIV-1-infected individuals obtained at different time intervals before and after the initiation of HAART were assessed for specific IFN-gamma, IL-2, IL-4, and IL-10 mRNA expression, using RT-PCR. Correlation with CD4+ T cell counts and viral load was also carried out. Before initiation of HAART, in all patients, little expression of specific IFN-gamma and IL-2 (type 1 cytokine) mRNA was noted. In contrast, expression of specific IL-4 and/or IL-10 (type 2) mRNA was readily detectable in the majority of patients. After initiation of HAART there was a continuous increase in IFN-gamma and IL-2 mRNA expression, although the latter occurred in lower amounts. This paralleled a dramatic reduction in viral load and increase in CD4+ T cell counts. Type 2 cytokine-specific mRNA expression fell to undetectable levels and in some cases reappeared later in the course of HAART. Predominant expression of type 2 cytokine mRNA, before initiation of HAART, concurs with previous findings of a dominant antiproliferative, type 2 cytokine profile during HIV-1 infection. Reversion of the cytokine profile, after HAART, to a strong type 1 profile suggests that in addition to suppressing virus replication directly the immune system may be given a chance to recover.