TAG HIV Basic Science, Vaccines, and Cure Project Blog

The Role of CD8 T Cells in Attenuated Vaccine Protection Against SIV

A paper from Chris Miller's research group at UC Davis reports that CD8 T cells can play a key role in protecting macaques against an intravaginal challenge with SIVmac239. The study employed a live-attenuated vaccine approach: macaques were immunized with an attenuated SIV/HIV hybrid (SHIV89.6) and then challenged 6-8 months later with the highly virulent SIVmac239. A subset of animals were depleted of CD8 T cells prior to challenge to explore the role of these responses in vaccine-mediated protection. At 14 days post-challenge, 9/9 controls and 4/12 immunized macaques were positive for SIV RNA in the plasma, and viral loads in the RNA-positive immunized animals were significantly lower than in the controls. All five immunized, CD8-depleted macaques showed detectable plasma SIV RNA and viral loads in these animals were significantly higher than in the other two groups.

The researchers also investigated the extent of post-challenge CD8 and CD4 T cell proliferation in the macaques, finding that expression of the Ki67 antigen remained stable in the immunized group but increased significantly in controls. Similarly, T cell apoptosis was increased in both controls and CD8-depleted animals but not in the immunized group. SIV-specific CD8 T cells were also significantly more polyfunctional and cytotoxic in both the blood and vagina of immunized macaques compared to the control and CD8-depleted groups. Notably, expansion of SIV-specific CD8 T cells occurred only in the vagina of vaccinees, and none of the five other compartments examined (including blood, lymph nodes and cervix). The researchers conclude that "the presence of SIV-specific CD8 T cells in the vagina on the day of vaginal SIV challenge and a modest expansion of effector T cells was sufficient to stop viral dissemination and uncontrolled SIV replication post-challenge." In discussing the results, they point out that these findings differ from those obtained with prime-boost vaccines, wherein SIV challenge typically leads to systemic proliferation of SIV-specific memory CD8 T cells, perhaps suggesting that induction of local effector CD8 T cells at the site of exposure should be the goal of T cell-based HIV vaccines.

JVI Accepts, published online ahead of print on 10 September 2008
J. Virol. doi:10.1128/JVI.01433-08

With minimal systemic T cell expansion, CD8+ T cells mediate protection from vaginal SIV challenge in rhesus macaques immunized with attenuated SHIV89.6

Meritxell Genescà, Pamela J. Skinner, Jung Joo Hong, Jun Li, Ding Lu, Michael B. McChesney, and Christopher J. Miller

Center for Comparative Medicine, California National Primate Research Center, University of California, Davis, Davis, CA, 95616 USA; Department of Veterinary and Biomedical Sciences, Department of Microbiology, University of Minnesota, St. Paul, MN, 55108 USA; Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine and Division of Infectious Diseases, School of Medicine, University of California, Davis, Davis, CA, 95616 USA

Abstract

The presence, at the time of challenge, of antiviral effector T cells in the vaginal mucosa of female rhesus macaques immunized with live-attenuated simian-human immunodeficiency virus (SHIV) 89.6 is associated with consistent and reproducible protection from pathogenic simian immunodeficiency virus (SIV) vaginal challenge (18). Here we definitively demonstrate the protective role of the SIV-specific CD8+ T cell response in the SHIV-immunized monkeys by CD8+ lymphocyte depletion, an intervention that abrogated SHIV-mediated control of challenge virus replication and largely eliminated the SIV-specific T cell responses in blood lymph nodes and genital mucosa. While in the T cell intact SHIV- immunized animals, polyfunctional and degranulating SIV-specific CD8+ T cells were present in the genital tract and lymphoid tissues from the day of challenge until day 14 post-challenge. Strikingly, expansion of SIV-specific CD8+ T cells in the immunized monkeys was minimal and limited to the vagina. Thus, protection from uncontrolled SIV replication in animals immunized with attenuated SHIV 89.6 is primarily mediated by CD8+ T cells that do not undergo dramatic systemic expansion after SIV challenge. These findings demonstrate that despite, and perhaps because of, minimal systemic expansion of T cells at the time of challenge, a stable population of effector-cytotoxic CD8+ T cells can provide significant protection from vaginal SIV challenge.

Microbial Translocation in Immunological Non-Responders to ART

A research letter in the journal AIDS reports that translocation of bacteria across the gut mucosa is associated with a failure to reconstitute CD4 T cells despite viral suppression by antiretroviral therapy (ART). The authors report that plasma levels of lipopolysaccharide (LPS), an indirect measure of microbial translocation, were significantly higher in both immunological non-responders (INRs) and individuals with untreated, advanced HIV infection compared to individuals on ART who experience good CD4 T cell reconstitution. The researchers also offer the first published direct evidence of microbial translocation in HIV infection by measuring bacterial 16s DNA in samples and then conducting sequencing experiments to show that the DNA was derived from gut bacteria species. Using this technique, they found that 16s DNA could be isolated from five out of seven INRs and six out of 12 individuals with advanced, untreated HIV infection but none of the seven individuals with a good CD4 T cell response to ART.

In discussing their results, the authors state: “by showing an association between LPS and bacterial DNA fragments in plasma, our study is the first to directly demonstrate bacterial translocation in both naïve and HAART-treated HIV-infected patients through the direct demonstration of enterobacteria genome sequences.” They also raise the issue of uncertainty regarding cause and effect, noting that microbial translocation may contribute to the lack of an immunological response to ART seen in INRs but also that, conversely, “bacterial translocation might be favoured in INRs by reduced T-cell-mediated competence failing to provide full immune control in mucosa and mesenteric lymph nodes, thus permitting peripheral egress and survival of bacteria.” In support of this possibility, they cite a paper from 1980 showing that microbial translocation occurs in athymic mice and can be reduced when the mice receive thymic grafts. The full text of this paper is available online. Interestingly, this is the same suggestion made by the recent murine study from Brigitta Stockinger’s group which found that reduce naïve T cell levels were associated with microbial translocation and immune activation (reported on the blog previously).

AIDS. 22(15):2035-2038, October 1, 2008.

Research Letters

Microbial translocation is associated with sustained failure in CD4+ T-cell reconstitution in HIV-infected patients on long-term highly active antiretroviral therapy.

Marchetti, Giulia; Bellistri, Giusi M; Borghi, Elisa; Tincati, Camilla; Ferramosca, Stefania; La Francesca, Maria; Morace, Giulia; Gori, Andrea; Monforte, Antonella d'Arminio

Abstract:

Patients with inefficient CD4+ T-cell recovery on virogically suppressive highly active antiretroviral therapy constitute a major clinical hurdle given the threat of HIV/AIDS disease progression. We show heightened circulating lipopolysaccharide associated with plasma enterobacterial DNA and highly activated Ki67+CD4+CD8+ in 24 immunologic-nonresponders (CD4+ T-cell <= 200; HIV-RNA <= 50) compared with 11 full responders (CD4+ T-cell >= 400; HIV-RNA <= 50). These data provide novel insight into INRs pathogenesis, since they correlate augmented systemic translocation of microbial bioproducts with T-cell hyperactivation.

Case Report Suggests Antiretroviral Prophylaxis May Have Attenuated HIV Infection

Several large trials are now underway exploring the efficacy of either tenofovir (Viread) or a tenofovir/emtricabine combination pill (Truvada) as pre-exposure prophylaxis (PrEP) to protect against HIV infection. Concerns have been raised regarding what might happen to people who become infected despite the drugs, particularly in terms of the development of resistance. A new case report just published in the Journal of AIDS by Nicole Prada and colleagues from the Aaron Diamond AIDS Research Center suggests that Truvada might have attenuated HIV infection in an individual receiving the drug for post-exposure prophylaxis (PEP), without causing resistance.

The individual involved was prescribed a four-week course of Truvada as PEP due to high risk sexual activity; this was then extended for two weeks after additional possible exposures were reported. After a three-week break, another four-week course was prescribed subsequent to additional exposures. HIV antibodies began to become detectable during the second course of PEP, but evolved very slowly over several months. The viral load at time of diagnosis was 213 copies and, after stopping the second course of PEP, viral load measured 647 copies and has remained around that level during follow-up. It is uncertain whether HIV infection was acquired prior to or during the first course of PEP; it is not thought to have occurred during the three week break from Truvada. Importantly, no mutations associated with resistance to either tenofovir or emtricabine could be detected. The researchers investigated whether the individual possessed any of the most common host factors known to be associated with slowed disease progression, including heterozygosity for the CCR5 delta32 mutation and possession of HLA B57 or B27; none of these factors were present (although the authors note that this was not an exhaustive analysis). The study authors suggest that this case report may offer hope that PrEP can attenuate HIV replication in individuals who experience breakthrough infections, conceivably impacting both the health of the individual and also reducing the possibility of onward transmission.

JAIDS Journal of Acquired Immune Deficiency Syndromes. September 2, 2008, Publish Ahead of Print

Drug-Susceptible HIV-1 Infection Despite Intermittent Fixed-Dose Combination Tenofovir/Emtricitabine as Prophylaxis Is Associated With Low-Level Viremia, Delayed Seroconversion, and an Attenuated Clinical Course.

Rapid Communication

Prada, Nicole PhD; Davis, Brandi BS; Jean-Pierre, Patrick MS; Roche, Matthew La BS; Duh, Fuh-Mei MS; Carrington, Mary PhD; Poles, Michael MD; Mehandru, Saurabh MD; Mohri, Hiroshi MD, PhD; Markowitz, Martin MD

Abstract:

Background: Continued high rates of HIV-1 transmission have fueled interest in the use of antiretrovirals to prevent infection. Attenuated infection with failure of tenofovir as prophylaxis has been reported in animal models. Here, we report a case of HIV-1 infection despite intermittent use of fixed-dose combination tenofovir and emtricitabine (FTC).

Methods: The patient was treated with tenofovir DF/FTC for reported repeated high-risk sexual exposures. After seroconversion, he was subjected to routine laboratory testing, CCR5 and HLA genotyping, and biopsy of gastrointestinal (GI) tissue. Resistance testing was performed both as bulk sequencing of plasma and cloning and sequencing of virus derived from plasma, peripheral blood mononuclear cells, and GI tissue.

Results: In this patient with no readily identifiable modifying host factors, acute HIV-1 infection with tenofovir DF/FTC-susceptible HIV-1 was associated with an attenuated clinical course, very low postseroconversion HIV-1 RNA levels, slow kinetics of seroconversion, and relative sparing of mucosal CD4+ T cells in the GI tract.

Conclusions: Despite the failure of tenofovir DF/FTC as prophylaxis, selection for drug-resistant transmission did not occur and the blunting of postinfection levels of viremia likely reduced the probability of subsequent forward transmissions during the acute phase. These results support continued investigations of the use of antiretrovirals as a means to reduce HIV-1 transmission.

A Note of Caution on siRNA Studies

A new paper in the “Instant Online” section of Human Gene Therapy offers a cautionary tale about interpreting results of studies employing short interfering RNA (siRNA) as a therapeutic strategy. The authors report that these siRNAs typically trigger interferon production while the control siRNA used in many studies (at least 13, by their count) has unusually weak immune-stimulating properties. They also show that the anti-influenza activity reported for some siRNAs is entirely dependent on immune stimulation; modification of these constructs to abrogate their capacity to induce interferon caused a complete loss of the anti-influenza effect. The authors close by stressing that “to avoid the potential of misinterpreting therapeutic efficacy caused by siRNA-mediated immune stimulation for a specific RNAi effect in animal models, it is critical for researchers to appreciate the capacity for siRNA to activate such a response and to fully characterize the immunostimulatory potential of both active and control siRNA sequences.”

Free full text PDF: http://www.liebertonline.com/doi/pdfplus/10.1089/hgt.2008.131

Hum Gene Ther. 2008 Aug 19. [Epub ahead of print]

Misinterpreting the therapeutic effects of siRNA caused by immune stimulation.

Robbins M, Judge A, Ambegia E, Choi C, Yaworski E, Palmer L, McClintock K, Maclachlan I.
Protiva Biotherapeutics Inc., Burnaby, British Columbia, Canada.

Activation of innate immunity has direct effects in modulating viral replication, tumor growth, angiogenesis, inflammatory and other immunological processes. It is now established that unmodified siRNA can activate this innate immune response and therefore there is real potential for siRNA to elicit non-specific therapeutic effects in a wide range of disease models. Here we demonstrate that in a murine model of Influenza infection, the anti-viral activity of siRNA is primarily due to immune stimulation elicited by the active siRNA duplexes and not the result of therapeutic RNAi as previously reported. We show that the misinterpretation stems from the use of a particular control GFP siRNA that we identify as having unusually low immunostimulatory activity compared to the active anti-influenza siRNA. Curiously, this GFP siRNA has served as a negative control for a surprising number of groups reporting therapeutic effects of siRNA. The inert immunologic profile of the GFP sequence was unique among a broad panel of published siRNA, all of which could elicit significant Interferon induction from primary immune cells. This panel included 8 active siRNA against viral, angiogenic and oncologic targets whose reported therapeutic efficacy was based on comparison to the non-immunostimulatory GFP siRNA. These results emphasize the need for researchers to anticipate, monitor and adequately control for siRNA-mediated immune stimulation and calls into question the interpretation of numerous published reports of therapeutic RNAi in vivo. The use of chemically modified siRNA with minimal immunostimulatory capacity will help to more accurately delineate the mechanism of action underlying such studies.

Dysfunctional CD8 T cells in HIV Infection: Helpless & Unhelpful

A number of studies have shown that TNF-related apoptosis-inducing ligand (TRAIL) levels are elevated in HIV infection, suggesting a possible role in pathogenesis. An interesting new paper now implicates CD8 T cells as contributing to TRAIL production in people with AIDS. The researchers, led by Stefanie Kuerten, note that TRAIL production by CD8 T cells has been observed in murine studies in which CD8 T cell responses are generated in the absence of CD4 T cell help. These reports led them to develop a novel ELISpot assay for antigen-specific TRAIL production in order to assess whether the murine findings were echoed in HIV infection, where CD4 T cell help is compromised.

Their results show that TRAIL production by HIV-specific CD8 T cells increases as CD4 counts fall; in their small cohort of individuals with HIV infection, TRAIL production was only detectable when peripheral blood CD4 T cell counts fell below 200. The researchers also found that TRAIL production increased as the capacity of the CD8 T cells to produce interferon-gamma was lost. The phenomenon appeared restricted to CD8 T cells primed in the absence of CD4 T cell help, as TRAIL production by CD8 T cells specific for EBV, CMV and flu was not observed. The authors note that future studies should investigate whether new CD8 T cell responses to antigens encountered subsequent to CD4 decline also show evidence of TRAIL production, as these data would predict. They also speculate that TRAIL production by CD8 T cells might exacerbate CD4 T cell depletion by inducing apoptosis (a known function of TRAIL).

PDF full text: http://www.liebertonline.com/doi/pdfplus/10.1089/aid.2008.0062

AIDS Res Hum Retroviruses. 2008 Aug 26. [Epub ahead of print]

The TRAIL of Helpless CD8(+) T Cells in HIV Infection.

Kuerten S, Asaad RJ, Schoenberger SP, Angelov DN, Lehmann PV, Tary-Lehmann M.
Department of Pathology, Case Western Reserve University, Cleveland, Ohio 44106., Institut I fuer Anatomie, University of Cologne, Cologne, Germany.

Abstract Our understanding of how CD4(+) T cells can regulate CD8(+) T cell responses in HIV infection is still incomplete. Recent evidence obtained in mice suggests that CD4(+) T cell help is required for efficient CD8(+) T cell-mediated immunity in chronic infection: CD8(+) T cells primed in the absence of such help release the TNF-related apoptosis-inducing ligand TRAIL and undergo apoptosis. Using a novel ELISPOT assay, in the present study we show that CD8(+) T cells are also a source of the antigen-specific TRAIL response in HIV-infected patients with CD4(+) T cell counts below 200. In patients with CD4(+) T cell counts above 200 TRAIL was not detectable. Accordingly, antigens to which patients have likely been exposed when CD4(+) T cell levels were high (e.g., influenza, CMV, and EBV) did not induce TRAIL. Within the HIV-positive donor population with low CD4(+) T cell counts a dissociation of the interferon-gamma (IFN-gamma) and TRAIL response to different HIV peptide epitopes was detectable suggesting impaired immunity to antigens that triggered TRAIL in the absence of IFN-gamma. Our findings emphasize that "helpless" CD8(+) T cells, i.e., cells that have been primed in the absence of CD4(+) T cell help, may play a crucial role in HIV infection. A "helpless" phenotype may impair CD8(+) T cell control of HIV and other infections and possibly contribute to the depletion of CD4(+) T cells via apoptosis. Immunizations and infections in this "helpless" state might result in ineffective CD8(+) T cell responses.

CD4 T Cell Review

Jinfang Zhu and William Paul review the current state of knowledge regarding the differentiation of CD4 T cell subsets, in honor of the 50th Anniversary of the American Society for Hematology (ASH). Access to the full text is free. This issue of Blood also contains a complementary and similarly detailed review addressing the development and function of B cells by Tucker LeBien and Thomas Tedder.

Blood, 1 September 2008, Vol. 112, No. 5, pp. 1557-1569.

ASH 50TH ANNIVERSARY REVIEW

CD4 T cells: fates, functions, and faults

Jinfang Zhu1, and William E. Paul1

1 Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD

Abstract

In 1986, Mosmann and Coffman identified 2 subsets of activated CD4 T cells, Th1 and Th2 cells, which differed from each other in their pattern of cytokine production and their functions. Our understanding of the importance of the distinct differentiated forms of CD4 T cells and of the mechanisms through which they achieve their differentiated state has greatly expanded over the past 2 decades. Today at least 4 distinct CD4 T-cell subsets have been shown to exist, Th1, Th2, Th17, and iTreg cells. Here we summarize much of what is known about the 4 subsets, including the history of their discovery, their unique cytokine products and related functions, their distinctive expression of cell surface receptors and their characteristic transcription factors, the regulation of their fate determination, and the consequences of their abnormal activation.

Blood, 1 September 2008, Vol. 112, No. 5, pp. 1570-1580.

ASH 50TH ANNIVERSARY REVIEW

B lymphocytes: how they develop and function

Tucker W. LeBien1, and Thomas F. Tedder2

1 Department of Laboratory Medicine/Pathology, University of Minnesota Cancer Center, Minneapolis; and 2 Department of Immunology, Duke University Medical Center, Durham, NC

Abstract

The discovery that lymphocyte subpopulations participate in distinct components of the immune response focused attention onto the origins and function of lymphocytes more than 40 years ago. Studies in the 1960s and 1970s demonstrated that B and T lymphocytes were responsible primarily for the basic functions of antibody production and cell-mediated immune responses, respectively. The decades that followed have witnessed a continuum of unfolding complexities in B-cell development, subsets, and function that could not have been predicted. Some of the landmark discoveries that led to our current understanding of B lymphocytes as the source of protective innate and adaptive antibodies are highlighted in this essay. The phenotypic and functional diversity of B lymphocytes, their regulatory roles independent of antibody production, and the molecular events that make this lineage unique are also considered. Finally, perturbations in B-cell development that give rise to certain types of congenital immunodeficiency, leukemia/lymphoma, and autoimmune disease are discussed in the context of normal B-cell development and selection. Despite the significant advances that have been made at the cellular and molecular levels, there is much more to learn, and cross-disciplinary studies in hematology and immunology will continue to pave the way for new discoveries.