Optimizing HIV-1-specific CD8+ T-cell induction by recombinant BCG in prime-boost regimens with heterologous viral vectors

The desire to induce HIV-1-specific responses soon after birth to prevent breast milk transmission of HIV-1 led us to propose a vaccine regimen which primes HIV-1-specific T cells using a recombinant Mycobacterium bovis bacillus Calmette-Guérin (rBCG) vaccine. Because attenuated live bacterial vaccines are typically not sufficiently immunogenic as stand-alone vaccines, rBCG-primed T cells will likely require boost immunization(s). Here, we compared modified Danish (AERAS-401) and Pasteur lysine auxotroph (222) strains of BCG expressing the immunogen HIVA for their potency to prime HIV-1-specific responses in adult BALB/c mice and examined four heterologous boosting HIVA vaccines for their immunogenic synergy. We found that both BCG.HIVA(401) and BCG.HIVA(222) primed HIV-1-specific CD8(+) T-cell-mediated responses. The strongest boosts were delivered by human adenovirus-vectored HAdV5.HIVA and sheep atadenovirus-vectored OAdV7.HIVA vaccines, followed by poxvirus MVA.HIVA; the weakest was plasmid pTH.HIVA DNA. The prime-boost regimens induced T cells capable of efficient in vivo killing of sensitized target cells. We also observed that the BCG.HIVA(401) and BCG.HIVA(222) vaccines have broadly similar immunologic properties, but display a number of differences mainly detected through distinct profiles of soluble intercellular signaling molecules produced by immune splenocytes in response to both HIV-1- and BCG-specific stimuli. These results encourage further development of the rBCG prime-boost regimen.     

Cytotoxic HIV-1 p55gag-specific CD4+ T cells produce HIV-inhibitory cytokines and chemokines

CD4⁺ T-helper cells appear to be essential in sustaining immune responses in chronic viral infections, as the maintenance of CD8⁺ cytotoxic T-lymphocyte responses and the control of viremia were demonstrated to depend on CD4⁺ T cell help. In order to investigate the function of HIV-specific CD4⁺ T cells in chronic HIV-1-infection, 49 chronically HIV-infected patients were analyzed before and 3 and 6 months after initiation of antiviral treatment. Ten patients showed a substantial, although weak, proliferative response to HIV-1-p55^gag protein for which no improvement was observed upon initiation of HAART. From one individual, HIV-1-p55^gag-specific CD4-positive T-cell clones were generated that were heterogeneous in their TCR V gene usage and HLA-DRB1*13 and DRB1*03 restricted, respectively. In addition, some CD4⁺ TCC produced substantial amounts of IFN- and MIP-1/, were perforin-positive, and showed cytotoxic activity. These diverse functional features of HIV-specific CD4⁺ T cells suggest that they may exert direct antiviral activity.     

CD4+ T-cell help amplifies innate signals for primary CD8+ T-cell immunity

CD8⁺ T cells provide an important component of protection against intracellular infections and cancer. Immune responses by these T cells involve a primary phase of effector expansion and differentiation, followed by a contraction phase leading to memory formation and, if antigen is re-encountered, a secondary expansion phase with more rapid differentiation. Both primary and secondary phases of CD8⁺ T-cell immunity have been shown to depend on CD4⁺ T-cell help, although during certain infections the primary phase is variable in this requirement. One explanation for such variability relates to the strength of associated inflammatory signals, with weak signals requiring help. Here, we focus on our studies that have dissected the requirements for help in the primary phase of the CTL response to herpes simplex virus, elucidating intricate interactions and communications between CD4⁺ T cells, various dendritic cell subsets, and CD8⁺ T cells. We place our studies in the context of others and describe a simple model of help where CD40 signaling amplifies innate signals to enable efficient CD8⁺ T-cell expansion and differentiation. This model facilitates CTL induction to various different agents, without altering the qualitative innate signals that direct other important arms of immunity.     

MHC class II-independent CD25+ CD4+ CD8alpha beta+ alpha beta T cells attenuate CD4+ T cell-induced transfer colitis

CD4+ alpha beta T cell populations that develop in mice deficient in MHC class II (through 'knockout' of either the Aalpha, or the Abeta chain of the I-A(b) molecule) comprise a major 'single-positive' (SP) CD4+ CD8- subset (60-90%) and a minor 'double-positive' (DP) CD4+ CD8alpha beta+ subset (10-40%). Many DP T cells found in spleen, mesenteric lymph nodes (MLN) and colonic lamina propria (cLP) express CD25, CD103 and Foxp3. Adoptive transfer of SP but not DP T cells from Aalpha(-/-) or Abeta(-/-) B6 mice into congenic RAG(-/-) hosts induces colitis. Transfer of SP T cells repopulates the host with only SP T cells; transfer of DP T cells repopulates the host with DP and SP T cells. Anti-CD25 antibody treatment of mice transplanted with DP T cells induces severe, lethal colitis; anti-CD25 antibody treatment of mice transplanted with SP T cells further aggravates the course of severe colitis. Hence, regulatory CD25+ T cells within (or developing from) the DP T cell population of MHC class II-deficient mice control the colitogenic potential of CD25- CD4+ T cells.     

Mucosal immunization of CD4+ T cell-deficient mice with an inactivated virus induces IgG and IgA responses in serum and mucosal secretions

Immunoglobulin (Ig) class switching can occur in the absence of alphabeta+ or gammadelta+ T cells when mice are infected with certain live viruses, although CD4 T helper cells are believed to be essential for induction of a high-affinity antibody response and for efficient isotype switching from IgM to IgG and IgA production. However, little information is available about the immune responses after mucosal immunization of CD4+ T cell-deficient mice with inactivated virus. In this study, we show that intranasal immunization with formalin-inactivated influenza A/PR8/34 virus induces IgG and IgA responses in serum and IgA responses in mucosal secretions in CD4+ T cell-deficient mice. All four subclasses of IgG were produced. IgG1/IgG2a ratios were found to be from 1 to 1.75, indicating that both Th1 and Th2 immune responses are induced by the inactivated influenza virus. The sera and mucosal secretions were found to have neutralizing activity against influenza virus in vitro. In addition, the mucosally immunized CD4+ T cell-deficient mice were protected completely from challenge with a lethal dose of live, pathogenic influenza virus. To our knowledge, this is the first demonstration that mucosal immunization with an inactivated virus induces immune responses in serum and mucosal secretions in CD4+ T cell-deficient mice.     

HIV-1 infection alters CD4+ memory T-cell phenotype at the site of disease in extrapulmonary tuberculosis

HIV-1-infected people have an increased risk of developing extrapulmonary tuberculosis (TB), the immunopathogenesis of which is poorly understood. Here, we conducted a detailed immunological analysis of human pericardial TB, to determine the effect of HIV-1 co-infection on the phenotype of Mycobacterium tuberculosis (MTB)-specific memory T cells and the role of polyfunctional T cells at the disease site, using cells from pericardial fluid and blood of 74 patients with (n = 50) and without (n = 24) HIV-1 co-infection. The MTB antigen-induced IFN-γ response was elevated at the disease site, irrespective of HIV-1 status or antigenic stimulant. However, the IFN-γ ELISpot showed no clear evidence of increased numbers of antigen-specific cells at the disease site except for ESAT-6 in HIV-1 uninfected individuals (p = 0.009). Flow cytometric analysis showed that CD4+ memory T cells in the pericardial fluid of HIV-1-infected patients were of a less differentiated phenotype, with the presence of polyfunctional CD4+ T cells expressing TNF, IL-2 and IFN-γ. These results indicate that HIV-1 infection results in altered phenotype and function of MTB-specific CD4+ T cells at the disease site, which may contribute to the increased risk of developing TB at all stages of HIV-1 infection.     

Immunisation with recombinant modified vaccinia virus Ankara expressing HIV-1 gag in HIV-1-infected subjects stimulates broad functional CD4+ T cell responses

Virus-specific CD4+ T cells with IL-2-secreting and/or proliferative capacity are detected readily in HIV-1-infected long-term nonprogressors and rarely in persons with untreated progressive infection. The contribution of these cells to viraemia control is uncertain, but this question might be addressed in clinical therapeutic vaccination studies. However, the quality of T helper responses induced by currently available HIV-1 vaccine candidates has not been explored in depth. We determined the effect of vaccination with modified vaccinia virus Ankara (MVA) expressing HIV-1 gag p24/p17 (MVA.HIVA) on HIV-1-specific CD4+ T cell responses in 16 chronically infected, highly active antiretroviral therapy (HAART)-treated subjects using CD8-depleted IFN-gamma ELISPOT assays, intracellular cytokine staining assays for IL-2 and IFN-gamma, and a CFSE-based proliferation assay. Gag-specific CD4+ T cell responses were significantly increased in magnitude and breadth after vaccination and targeted both known and new epitopes, several of which were also recognised by healthy HIV-uninfected volunteers immunised with the same vaccines. The frequencies of CD4+ T cells expressing IL-2 or IFN-gamma, alone or simultaneously, were also augmented. These findings indicate that functional virus-specific T helper cells can be boosted by vaccination in chronic HIV-1 infection. Further evaluation of their role in viraemia control is warranted.     

Type I interferon supports primary CD8+ T-cell responses to peptide-pulsed dendritic cells in the absence of CD4+ T-cell help

CD8(+) T-cell responses to non-pathogen, cell-associated antigens such as minor alloantigens or peptide-pulsed dendritic cells (DC) are usually strongly dependent on help from CD4(+) T cells. However, some studies have described help-independent primary CD8(+) T-cell responses to cell-associated antigens, using immunization strategies likely to trigger natural killer (NK) cell activation and inflammatory cytokine production. We asked whether NK cell activation by MHC I-deficient cells, or administration of inflammatory cytokines, could support CD4(+) T-cell help-independent primary responses to peptide-pulsed DC. Injection of MHC I-deficient cells cross-primed CD8(+) T-cell responses to the protein antigen ovalbumin (OVA) and the male antigen HY, but did not stimulate CD8(+) T-cell responses in CD4-depleted mice; hence NK cell stimulation by MHC I-deficient cells did not replace CD4(+) T-cell help in our experiments. Dendritic cells cultured with tumour necrosis factor-α (TNF-α) or type I interferon-α (IFN-α) also failed to prime CD8(+) T-cell responses in the absence of help. Injection of TNF-α increased lymph node cellularity, but did not generate help-independent CD8(+) T-cell responses. In contrast, CD4-depleted mice injected with IFN-α made substantial primary CD8(+) T-cell responses to peptide-pulsed DC. Mice deficient for the type I IFN receptor (IFNR1) made CD8(+) T-cell responses to IFNR1-deficient, peptide-pulsed DC; hence IFN-α does not appear to be a downstream mediator of CD4(+) T-cell help. We suggest that primary CD8(+) T-cell responses will become help-independent whenever endogenous IFN-α secretion is stimulated by tissue damage, infection, or autoimmune disease.     

Robust CD4+ and CD8+ T cell responses to SIV using mRNA-transfected DC expressing autologous viral Ag

A potentially powerful strategy for therapeutic HIV vaccination is the use of DC transfected with mRNA encoding autologous viral Ag, as epitopes presented by transfected DC would exactly reflect those expressed by infected cells in the individual. Using human and rhesus macaque monocyte-derived DC, we show that nucleofection is a superior method for mRNA transfection, resulting in high-level protein expression and DC maturation. DC transfected with SIV gag isolated from an infected monkey stimulated robust Ag-specific recall T cell responses of similar magnitude to those induced by peptide-pulsed PBMC that were predominantly CD8+ T cell mediated. Enhanced CD4+ T cell responses were stimulated when Gag was redirected into the lysosomal pathway via the targeting signal derived from lysosome-associated membrane protein-1 (LAMP-1). Rhesus DC transfected with lysosome-targeted gag encoding an escape mutation in an immunodominant CTL epitope stimulated CD4+ and CD8+ T cell responses of almost equivalent magnitude directed towards undefined epitopes outside of the mutated region. Finally, gag-transfected DC from SIV-infected monkeys stimulated significant Ag-specific recall T cell responses in an entirely autologous system. These findings demonstrate that mRNA-transfected DC expressing SIV Ag derived from infected monkeys stimulate broad and relevant T cell responses, supporting this approach for therapeutic HIV vaccine development.     

Co-expression of CD8α in CD4+ T cell receptor αβ+ T cells migrating into the murine small intestine epithelial layer

We investigated the surface phenotype of CD3⁺CD4⁺ T cell receptor (TCR) αβ⁺ T cells repopulating the intestinal lymphoid tissues of C.B-17 scidlscid (severe-combined immunodeficient; scid) (H-2^d, L^d+) mice. CD4⁺ CD8^? T cells were cell sorter-purified from various secondary and tertiary lymphoid organs of congenic C.B-17 +/+ (H-2^d, L^d+) or semi-syngeneic dm2 (H-2^d, L^d?) immunocompetent donor mice. After transfer of 10⁵ cells into young scid mice, a mucosa-homing, memory CD44^hi CD45RB^lo CD4⁺ T cell population was selectively engrafted. Large numbers of single-positive (SP) CD3⁺ CD2⁺ CD28⁺ CD4⁺ CD^8? T cells that expressed the α₄ integrin chain CD49d were found in the spleen, the mesenteric lymph nodes, the peritoneal cavity and the gut lamina propria of transplanted scid mice. Unexpectedly, large populations of donor-type doublepositive (DP) CD4⁺ CD8α⁺ CD8β^? T cells with high expression of the CD3/TCR complex appeared in the epithelial layer of the small intestine of transplanted scid mice. In contrast to SP CD4⁺ T cells, the intraepithelial DP T cells showed low expression of the CD2 and the CD28 co-stimulator molecules, and of the α₄ integrin chain CD49d, but expressed high levels of the α_IEL integrin chain CD103. The TCR-Vβ repertoire of DP but not SP intraepithelial CD4⁺ T cells was biased towards usage of the Vβ6 and Vβ8 viable domains. Highly purified populations of SP and DP CD4⁺ T cell populations from the small intestine epithelial layer of transplanted scid mice had different abilities to repopulate secondary scid recipient mice: SP CD4⁺ T cells repopulated various lymphoid tissues of the immunodeficient host, while intraepithelial DP CD4⁺ T cells did not. Hence, a subset of CD3⁺ CD4⁺ TCRαβ⁺ T cells apparently undergoes striking phenotypic changes when it enters the microenvironment of the small intestine epithelial layer.     

Virtual memory CD8+ T cells restrain the viral reservoir in HIV-1-infected patients with antiretroviral therapy through derepressing KIR-mediated inhibition

The viral reservoir is the major hurdle in developing and establishing an HIV cure. Understanding factors affecting the size and decay of this reservoir is crucial for the development of therapeutic strategies. Recent work highlighted that CD8+ T cells are involved in the control of viral replication in ART-treated HIV-1-infected individuals, but how CD8+ T cells sense and restrict the HIV reservoir are not fully understood. Here, we demonstrate that a population of unconventional CD45RA+, PanKIR+, and/or NKG2A+ virtual memory CD8+ T cells (T_VM cells), which confer rapid and robust protective immunity against pathogens, plays an important role in restraining the HIV DNA reservoir in HIV-1-infected patients with effective ART. In patients undergoing ART, T_VM cells negatively correlate with HIV DNA and positively correlate with circulating IFN-α2 and IL-15. Moreover, T_VM cells constitutively express high levels of cytotoxic granule components, including granzyme B, perforin and granulysin, and demonstrate the capability to control HIV replication through both cytolytic and noncytolytic mechanisms. Furthermore, by using an ex vivo system, we showed that HIV reactivation is effectively suppressed by T_VM cells through KIR-mediated recognition. This study suggests that T_VM cells are a promising target to predict posttreatment virological control and to design immune-based interventions to reduce the reservoir size in ART-treated HIV-1-infected individuals.     

Critical role for IL-21 in both primary and memory anti-viral CD8+ T-cell responses

While it is well established that CD8(+) T cells generated in the absence of CD4(+) T cells mediate defective recall responses, the mechanism by which CD4(+) T cells confer help in the generation of CD8(+) T-cell responses remains poorly understood. To determine whether CD4(+) T-cell-derived IL-21 is an important regulator of CD8(+) T-cell responses in help-dependent and -independent viral infections, we examined these responses in the IL-21Rα(-/-) mouse model. We show that IL-21 has a role in primary CD8(+) T-cell responses and in recall CD8(+) T-cell responses in help-dependent viral infections. This effect is due to a direct action of IL-21 in enhancing the proliferation of virus-specific CD8(+) T cells and reducing their TRAIL expression. These findings indicate that IL-21 is an important mediator of CD4(+) T-cell help to CD8(+) T cells.     

T-cell homeostasis in HIV-1 infection

Failure of T-cell homeostasis is an important feature of HIV-1 infection. Substantial evidence indicates that T-cell homeostasis is independent of CD4⁺and CD8⁺subsets, and this may contribute to the decline of CD4⁺T cells to low levels in this disease. Moreover, failure of T-cell homeostasis appears to precede the development of clinically-defined AIDS by approximately 1.5 to 2 years and is thus an important milestone in HIV-1 disease progression. We argue that T-cell turnover and depletion of memory cells in HIV-1 infection can be viewed as the reverse of the process by which immune reconstitution occurs after stem cell transplantation, and that changes in the functional level of T-cell memory may be critical to both processes. An understanding of the relationship between T-cell memory and regeneration of lost T cells may help preserve and/or reconstitute immune system homeostasis in HIV-1-infected individuals.     

CD8+ suppressor-mediated regulation of human CD4+ T cell responses to glutamic acid decarboxylase 65

Although potentially autoreactive T cells are present even in healthy subjects, most individuals do not develop autoimmune disease. It has been well demonstrated that CD4+ CD25+ regulatory T cells play a significant role in controlling the expansion of autoreactive T cells in the periphery. However, some healthy individuals exhibit measurable responses to self peptide even in the presence of CD4+ CD25+ regulatory cells. This article describes the regulation of human CD4+ T cell responses to glutamic acid decarboxylase 65 (GAD65), an autoantigen implicated in type-1 diabetes, by autologous CD8+ suppressor T cells. In cells cultured from healthy individuals, the inclusion of autologous CD8+ T cells at physiological levels resulted in a dramatic decrease in the magnitude of in vitro CD4+ T cell responses to GAD65 peptide. Based on transwell experiments, the observed suppression was cell contact-dependent. However, antibody blocking studies indicated that suppression was mediated by IL-10. Cell fractionation studies suggested that CD8+ suppressor T cells originate from the CD45RA+ CD27- population. The suppression of CD4+ T cell responses to GAD65 in healthy individuals raises the possibility that CD8+ suppressor T cells play an important role in controlling potentially autoreactive T cells in the general population.     

Direct measurement of CD4+ and CD8+ T-cell responses to CMV in HIV-1-infected subjects

Data from murine models of chronic viral infection suggest that CD4+ T-cell responses to viral pathogens are important in sustaining the number and/or function of CD8+ cytotoxic T-cell (CTL) effectors. In this study, we used cytokine flow cytometry (CFC), staining with HLA-A*0201-peptide tetramers, and peptide stimulation with epitopic peptides to study functional CD4+ and CD8+ T-cell responses to cytomegalovirus (CMV) in human subjects coinfected with CMV and the human immunodeficiency virus, type 1 (HIV-1). We show that strong CD4+ and CD8+ T-cell responses to CMV antigens are sustained over time in HIV-1-infected individuals. Those who maintain a strong CD4+ T-cell response to CMV are also likely to maintain higher frequencies of CD8+ T cells capable of binding to HLA-A*0201-CMV pp65 (A2-pp65) tetramers as well as responses to pp65 peptide stimulation with effector cytokine production. These data support the hypothesis that declines in frequencies of CD4+ T-cell responses to CMV are associated with an inability to sustain high levels of CMV-specific CD8+ T-cell responses in HIV-1-infected subjects. These declines may precede the onset of CMV-associated end organ disease.     

Correlation between gag-specific CD8 T-cell responses, viral load, and CD4 count in HIV-1 infection is dependent on disease status

BACKGROUND: It still remains controversial which kind of relationships exist between HIV-1-specific CD8 T-cell responses and HIV RNA load or CD4 count over the course of the infection. This study was designed to investigate the role of HIV-specific CD8 responses in patients with different disease status. METHODS: Three cohorts of patients were selected according to CD4 count levels: long-term nonprogressors (LTNPs, n = 19), asymptomatic progressors (CD4 counts between 500 and 350 cells/mm(3), n = 14), and progressors (CD4 counts <350 cells/mm(3), n = 23). Six of the LTNPs experiencing a quick loss of CD4 T-cells and another 6 LTNPs with stable CD4 counts were followed up. T-cell responses were studied using interferon (IFN) gamma-ELISpot assay against HIV p24 and 11 pools of HIV-Gag peptides. RESULTS: No significant differences were found in Gag-specific CD8 responses among the 3 cohorts. However, inverse correlations were identified between CD8 responses and CD4 counts in asymptomatic progressors and between CD4 responses and viral loads in progressors. In addition, the sequential dynamics of CD8 responses in 6 LTNPs showed that with a quick loss of CD4 T-cells around the range of 500 to 300 cells/mm(3), more vigorous CD8 responses were induced simultaneously, and plasma viremia was still kept relatively stable. CONCLUSIONS: These data suggest that the relationship between CD8 response and viral load or CD4 count is not universally consistent throughout the entire course of HIV-1 infection. Gag-specific CD8 responses may play differential roles in different stages of HIV-1 infection, and the maintenance of a threshold level of CD4 T-cells may contribute to mediate effective HIV-specific responses in natural control of HIV-1 infection.