Yvette Edghill-Smith

Impairment of Gag-Specific CD8+ T-Cell Function in Mucosal and Systemic Compartments of Simian Immunodeficiency Virus mac251- and Simian-Human Immunodeficiency Virus KU2-Infected Macaques

ABSTRACT The identification of several simian immunodeficiency virus mac251 (SIVmac251) cytotoxic T-lymphocyte epitopes recognized by CD8+ T cells of infected rhesus macaques carrying the Mamu-A*01 molecule and the use of peptide-major histocompatibility complex tetrameric complexes enable the study of the frequency, breadth, functionality, and distribution of virus-specific CD8+ T cells in the body. To begin to address these issues, we have performed a pilot study to measure the virus-specific CD8+ and CD4+ T-cell response in the blood, lymph nodes, spleen, and gastrointestinal lymphoid tissues of eight Mamu-A*01-positive macaques, six of those infected with SIVmac251 and two infected with the pathogenic simian-human immunodeficiency virus KU2. We focused on the analysis of the response to peptide p11C, C-M (Gag 181), since it was predominant in most tissues of all macaques. Five macaques restricted viral replication effectively, whereas the remaining three failed to control viremia and experienced a progressive loss of CD4+ T cells. The frequency of the Gag 181 (p11C, C→M) immunodominant response varied among different tissues of the same animal and in the same tissues from different animals. We found that the functionality of this virus-specific CD8+ T-cell population could not be assumed based on the ability to specifically bind to the Gag 181 tetramer, particularly in the mucosal tissues of some of the macaques infected by SIVmac251 that were progressing to disease. Overall, the functionality of CD8+ tetramer-binding T cells in tissues assessed by either measurement of cytolytic activity or the ability of these cells to produce gamma interferon or tumor necrosis factor alpha was low and was even lower in the mucosal tissue than in blood or spleen of some SIVmac251-infected animals that failed to control viremia. The data obtained in this pilot study lead to the hypothesis that disease progression may be associated with loss of virus-specific CD8+ T-cell function.

Systemic Immunization with an ALVAC-HIV-1/Protein Boost Vaccine Strategy Protects Rhesus Macaques from CD4+ T-Cell Loss and Reduces both Systemic and Mucosal Simian-Human Immunodeficiency Virus SHIVKU2 RNA Levels

ABSTRACT Transmission of human immunodeficiency virus type 1 (HIV-1) occurs primarily via the mucosal route, suggesting that HIV-1 vaccines may need to elicit mucosal immune responses. Here, we investigated the immunogenicity and relative efficacy of systemic immunization with two human ALVAC-HIV-1 recombinant vaccines expressing Gag, Pol, and gp120 (vCP250) or Gag, Pol, and gp160 (vCP1420) in a prime-boost protocol with their homologous vaccine native Env proteins. The relative efficacy was measured against a high-dose mucosal exposure to the pathogenic neutralization-resistant variant SHIVKU2 (simian-human immunodeficiency virus). Systemic immunization with both vaccine regimens decreased viral load levels not only in blood but unexpectedly also in mucosal sites and protected macaques from peripheral CD4+ T-cell loss. This protective effect was stronger when the gp120 antigen was included in the vaccine. Inclusion of recombinant Tat protein in the boosting phase along with the Env protein did not contribute further to the preservation of CD4+ T cells. Thus, systemic immunization with ALVAC-HIV-1 vaccine candidates elicits anti-HIV-1 immune responses able to contain virus replication also at mucosal sites in macaques.

Boosting of SIV-specific immune responses in rhesus macaques by repeated administration of Ad5hr-SIVenv/rev and Ad5hr-SIVgag recombinants.

Previous non-human primate studies have shown replication competent adenovirus (Ad) HIVenv/rev and SIVenv/rev recombinants to be promising vaccine candidates. To broaden induced immunity in rhesus macaques, an Ad type 5 host range (Ad5hr) mutant vector with an inserted SIV gag gene was added to the vaccine regimen. Immunity to the encoded SIV Env, Rev, and Gag gene products was evaluated following two immunizations with the same recombinants. The vaccines were administered intranasally plus orally via stomach tube at weeks 0 and 12. The recombinants replicated well in the upper respiratory tract but poorly in the gut, suggesting enteric-coated capsules might improve oral delivery to the intestine. SIV-specific cellular immunity was induced in all 16 immunized macaques. Fourteen exhibited positive interferon-gamma (IFN-gamma) ELISPOT responses, and nine, including two lacking IFN-gamma responses, exhibited SIV-specific T-cell proliferative activity. IFN-gamma secreting peripheral blood mononuclear cells (PBMCs) in response to SIV Gag, Env, and Rev peptides were induced in 73, 53, and 27% of macaques, respectively, and were boosted two- to four-fold by the second immunization. A persistent response to Gag was evident at least 10 weeks thereafter. p11C tetramer staining confirmed elicitation of SIV Gag-specific CD8+ T-cells in Mamu-A*01 macaques. Proliferative responses were more frequent after the second immunization, and binding antibody titers to SIV gp120 were significantly boosted by the immunization regimen. We conclude that a second administration of recombinants based in the same Ad5hr vector can effectively boost immunity to inserted gene products, obviating development of several recombinants in different Ad serotypes for multiple immunizations.

Contrasting Effects of Low-Dose IL-2 on Vaccine-Boosted Simian Immunodeficiency Virus (SIV)-Specific CD4+ and CD8+ T Cells in Macaques Chronically Infected with SIVmac251

IL-2, the first cytokine discovered with T cell growth factor activity, is now known to have pleiotropic effects on T cells. For example, it can promote growth, survival, and differentiation of Ag-selected cells, or facilitate Ag-induced cell death of T cells when Ag persists, and in vivo, it is thought to contribute to the regulation of the size of adaptive T cell response. IL-2 is deficient in HIV-1 infection and has been used in the management of HIV-1-infected individuals undergoing antiretroviral therapy. In this study, we investigated how continuous low-dose IL-2 affected the CD4+ and CD8+ T cell response induced by two inoculations of a canarypox recombinant SIV-based vaccine candidate in healthy macaques chronically infected with SIVmac251. These macaques had normal levels of CD4+ T cells at the beginning of antiretroviral therapy treatment. Vaccination in the presence of IL-2 significantly augmented Gag-specific CD8+ T cell responses, but actually reduced Gag-specific CD4+ T cell responses. Although IL-2 at low doses did not change the overall concentration of circulating CD4+ or CD8+ T cells, it expanded the frequency of CD4+CD25+ T cells. Depletion of the CD4+CD25+ T cells in vitro, however, did not result in a reconstitution of Gag-specific CD4+ responses or augmentation of SIV-specific CD8+ T cell responses. Thus, we conclude that the decrease in virus-specific CD4+ T cell response may be due to IL-2-promoted redistribution of cells from the circulation, or due to Ag-induced cell death, rather than suppression by a T regulatory population.

Prior DNA immunization enhances immune response to dominant and subdominant viral epitopes induced by a fowlpox-based SIVmac vaccine in long-term slow-progressor macaques infected with SIVmac251

A therapeutic vaccine for individuals infected with HIV-1 and treated with antiretroviral therapy (ART) should be able to replenish virus-specific CD4+ T-cells and broaden the virus-specific CD8+ T-cell response in order to maintain CD8+ T-cell function and minimize viral immune escape after ART cessation. Because a combination of DNA and recombinant poxvirus vaccine modalities induces high levels of virus-specific CD4+ T-cell response and broadens the cytolytic activity in naive macaques, we investigated whether the same results could be obtained in SIVmac251-infected macaques. The macaques studied here were long-term nonprogressors that naturally contained viremia but were nevertheless treated with a combination of antiviral drugs to assess more carefully the effect of vaccination in the context of ART. The combination of a DNA expressing the gag and pol genes (DNA-SIV-gp) of SIVmac239 followed by a recombinant fowlpox expressing the same SIVmac genes (FP-SIV-gp) was significantly more immunogenic than two immunizations of FP-SIV-gp in SIVmac251-infected macaques treated with ART. The DNA/FP combination significantly expanded and broadened Gag-specific T-cell responses measured by tetramer staining, ELISPOT, and intracellular cytokine staining and measurement of ex vivo cytolytic function. Importantly, the combination of these vaccine modalities also induced a sizeable expansion in most macaques of Gag-specific CD8-(CD4+) T-cells able to produce TNF-alpha. Hopefully, this modality of vaccine combination may be useful in the clinical management of HIV-1-infected individuals.

Rhesus Macaque Resistance to Mucosal Simian Immunodeficiency Virus Infection Is Associated with a Postentry Block in Viral Replication

ABSTRACT Elucidation of the host factors which influence susceptibility to human immunodeficiency virus or simian immunodeficiency virus (SIV) infection and disease progression has important theoretical and practical implications. Rhesus macaque 359, a vaccine control animal, resisted two successive intravaginal challenges with SIVmac251 and failed to seroconvert. Here, after an additional intrarectal SIVmac32H challenge, macaque 359 remained highly resistant to infection. Viral RNA (106 copies/ml) was observed in plasma only at week 2 postchallenge. Virus isolation and proviral DNA were positive only once at week eight postchallenge. The animal remained seronegative and cleared SIV in vivo. Its blood and lymph node cells obtained at 49 weeks after intrarectal challenge did not transmit SIV to a naive macaque. We found that the resistance of macaque 359 to SIV infection was not due to a high level of CD8+ suppressor activity but to an inherent resistance of its CD4+ T cells. To elucidate the basis for the unusually strong resistance of macaque 359 to SIV infection in vivo and in vitro, we investigated early events of viral infection and replication in CD4+ cells of macaque 359, including expression and mutation screening of SIV coreceptors and analysis of viral entry and reverse transcription. Mutation screening revealed no genetic alteration in SIV coreceptors. PCR analysis revealed a significant delay in production of early in vitro reverse transcription intermediates in macaque 359 cells compared to susceptible controls, but cell fusion assays showed that SIV entered the CD4+ CCR5+ cells of macaque 359 as readily as cells of macaques susceptible to SIV infection. Our results suggest that the resistance of macaque 359 to SIV infection is due to a postentry block in viral replication and implicate a cellular inhibitory mechanism in its CD4+ T cells. Identification of this host mechanism will help further elucidate the biochemistry of reverse transcription and may suggest therapeutic strategies. Determining the prevalence of this host resistance mechanism among macaques may lead to better design of SIV pathogenesis and vaccine studies.

Effects of intestinal survival surgery on systemic and mucosal immune responses in SIV‐infected rhesus macaques

Abstract: Evaluation of cellular immunity in the intestinal lamina propria of rhesus macaques has been used previously to assess protective immunity against mucosal simian immunodeficiency virus (SIV) challenges. As this technique requires survival surgery to obtain jejunal tissue, effects of surgical stress on the immune system were investigated. SIV‐specific immune responses, including IgG and IgA binding antibodies in sera and mucosal secretions, IgG and IgA secreting cells in peripheral blood, IgG neutralizing antibodies, T‐cell proliferative responses, and interferon‐γ secretion by peripheral blood mononuclear cells, were evaluated pre‐ and post‐surgery in macaques immunized with adenovirus‐SIV recombinant vaccines and SIV envelope protein and in SIV‐infected macaques. No differences in these immune parameters were observed in SIV‐naïve, immunized macaques or healthy SIV‐infected macaques with regard to surgery. A dramatic increase in total IgA antibody level following surgery in the rectal secretions of one SIV‐infected macaque that was rapidly progressing to AIDS and failed to recover from surgery was attributed to an abscess that developed at the intestinal site. To date, nearly 30 other macaques have undergone the intestinal survival surgery, some on more than one occasion, without experiencing any clinical difficulty. Overall, our results suggest that in healthy macaques, intestinal resection survival surgery can be conducted safely. Further, the method can be used to reliably sample the intestinal mucosa without major or persistent impact on humoral or cellular immune responses.