Design and Evaluation of Optimized Artificial HIV-1 Poly-T Cell-Epitope Immunogens

A successful HIV vaccine in addition to induction of antibody responses should elicit effective T cell responses. Here we described possible strategies for rational design of T-cell vaccine capable to induce high levels of both CD4+ and CD8+ T- cell responses. We developed artificial HIV-1 polyepitope T-cell immunogens based on the conserved natural CD8+ and CD4+ T cell epitopes from different HIV-1 strains and restricted by the most frequent major human leukocyte antigen (HLA) alleles. Designed immunogens contain optimized core polyepitope sequence and additional “signal” sequences which increase epitope processing and presentation to CD8+ and CD4+ T-lymphocytes: N-terminal ubiquitin, N-terminal signal peptide and C-terminal tyrosine motif of LAMP-1 protein. As a result we engineered three T cell immunogens – TCI-N, TCI-N2, and TCI-N3, with different combinations of signal sequences. All designed immunogens were able to elicit HIV-specific CD4+ and CD8+ T cell responses following immunization. Attachment of either ubiquitin or ER-signal/LAMP-1 sequences increased both CD4+ and CD8+ mediated HIV-specific T cell responses in comparison with polyepitope immunogen without any additional signal sequences. Moreover, TCI-N3 polyepitope immunogen with ubiquitin generated highest magnitude of HIV-specific CD4+ and CD8+ T cell responses in our study. Obtained data suggests that attachment of signal sequences targeting polyepitope immunogens to either MHC class I or MHC class II presentation pathways may improve immunogenicity of T-cell vaccines. These results support the strategy of the rational T cell immunogen design and contribute to the development of effective HIV-1 vaccine.     

ALVAC-SIV-gag-pol-env-based vaccination and macaque major histocompatibility complex class I (A*01) delay simian immunodeficiency virus SIVmac-induced immunodeficiency.

T-cell-mediated immune effector mechanisms play an important role in the containment of human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) replication after infection. Both vaccination- and infection-induced T-cell responses are dependent on the host major histocompatibility complex classes I and II (MHC-I and MHC-II) antigens. Here we report that both inherent, host-dependent immune responses to SIVmac251 infection and vaccination-induced immune responses to viral antigens were able to reduce virus replication and/or CD4+ T-cell loss. Both the presence of the MHC-I Mamu-A*01 genotype and vaccination of rhesus macaques with ALVAC-SIV-gag-pol-env (ALVAC-SIV-gpe) contributed to the restriction of SIVmac251 replication during primary infection, preservation of CD4+ T cells, and delayed disease progression following intrarectal challenge exposure of the animals to SIV(mac251 (561)). ALVAC-SIV-gpe immunization induced cytotoxic T-lymphocyte (CTL) responses cumulatively in 67% of the immunized animals. Following viral challenge, a significant secondary virus-specific CD8+ T-cell response was observed in the vaccinated macaques. In the same immunized macaques, a decrease in virus load during primary infection (P = 0.0078) and protection from CD4 loss during both acute and chronic phases of infection (P = 0.0099 and P = 0.03, respectively) were observed. A trend for enhanced survival of the vaccinated macaques was also observed. Neither boosting the ALVAC-SIV-gpe with gp120 immunizations nor administering the vaccine by the combination of mucosal and systemic immunization routes increased significantly the protective effect of the ALVAC-SIV-gpe vaccine. While assessing the role of MHC-I Mamu-A*01 alone in the restriction of viremia following challenge of nonvaccinated animals with other SIV isolates, we observed that the virus load was not significantly lower in Mamu-A*01-positive macaques following intravenous challenge with either SIV(mac251 (561)) or SIV(SME660). However, a significant delay in CD4+ T-cell loss was observed in Mamu-A*01-positive macaques in each group. Of interest, in the case of intravenous or intrarectal challenge with the chimeric SIV/HIV strains SHIV(89.6P) or SHIV(KU2), respectively, MHC-I Mamu-A*01-positive macaques did not significantly restrict primary viremia. The finding of the protective effect of the Mamu-A*01 molecule parallels the protective effect of the B*5701 HLA allele in HIV-1-infected humans and needs to be accounted for in the evaluation of vaccine efficacy against SIV challenge models.     

CD8 T-Cells from Most HIV-Infected Patients Lack Ex Vivo HIV-Suppressive Capacity during Acute and Early Infection

The strong CD8+ T-cell-mediated HIV-1-suppressive capacity found in a minority of HIV-infected patients in chronic infection is associated with spontaneous control of viremia. However, it is still unclear whether such capacities were also present earlier in the CD8+ T cells from non controller patients and then lost as a consequence of uncontrolled viral replication. We studied 50 patients with primary HIV-1-infection to determine whether strong CD8+ T-cell-mediated HIV suppression is more often observed at that time. Despite high frequencies of polyfunctional HIV-specific CD8+ T-cells and a strong CD4+ T-helper response, CD8+ T-cells from 48 patients lacked strong HIV-suppressive capacities ex vivo. This indicates that the superior HIV-suppressive capacity of CD8+ T-cells from HIV controllers is not a general characteristic of the HIV-specific CD8+ T cell response in primary HIV infection.     

Induction of human immunodeficiency virus type 1 (HIV-1)-specific T-cell responses in HIV vaccine trial participants who subsequently acquire HIV-1 infection

Candidate human immunodeficiency virus type 1 (HIV-1) vaccines designed to elicit T-cell immunity in HIV-1-uninfected persons are under investigation in phase I to III clinical trials. Little is known about how these vaccines impact the immunologic response postinfection in persons who break through despite vaccination. Here, we describe the first comprehensive characterization of HIV-specific T-cell immunity in vaccine study participants following breakthrough HIV-1 infection in comparison to 16 nonvaccinated subjects with primary HIV-1 infection. Whereas none of the 16 breakthrough infections possessed vaccine-induced HIV-1-specific T-cell responses preinfection, 85% of vaccinees and 86% of nonvaccinees with primary HIV-1 infection developed HIV-specific T-cell responses postinfection. Breakthrough subjects' T cells recognized 43 unique HIV-1 T-cell epitopes, of which 8 are newly described, and 25% were present in the vaccine. The frequencies of gamma interferon (IFN-gamma)-secreting cells recognizing epitopes within gene products that were and were not encoded by the vaccine were not different (P = 0.64), which suggests that responses were not anamnestic. Epitopes within Nef and Gag proteins were the most commonly recognized in both vaccinated and nonvaccinated infected subjects. One individual controlled viral replication without antiretroviral therapy and, notably, mounted a novel HIV-specific HLA-C14-restricted Gag LYNTVATL-specific T-cell response. Longitudinally, HIV-specific T cells in this individual were able to secrete IFN-gamma and tumor necrosis factor alpha, as well as proliferate and degranulate in response to their cognate antigenic peptides up to 5 years postinfection. In conclusion, a vaccinee's ability to mount an HIV-specific T-cell response postinfection is not compromised by previous immunization, since the CD8+ T-cell responses postinfection are similar to those seen in vaccine-na?ve individuals. Finding an individual who is controlling infection highlights the importance of comprehensive studies of breakthrough infections in vaccine trials to determine whether host genetics/immune responses and/or viral characteristics are responsible for controlling viral replication.     

CD8 T-cell recognition of multiple epitopes within specific Gag regions is associated with maintenance of a low steady-state viremia in human immunodeficiency virus type 1-seropositive patients

The importance of HLA class I-restricted CD8 T-cell responses in the control of human immunodeficiency virus (HIV) infection is generally accepted. While several studies have shown an association of certain HLA class I alleles with slower disease progression, it is not fully established whether this effect is mediated by HIV-specific CD8 T-cell responses restricted by these alleles. In order to study the influence of the HLA class I alleles on the HIV-specific CD8 T-cell response and on viral control, we have assessed HIV-specific epitope recognition, plasma viral load, and expression of HLA class I alleles in a cohort of HIV-seropositive bar workers. Possession of the HLA class I alleles B5801, B8101, and B0702 was associated with a low median viral load and simultaneously with a broader median recognition of Gag epitopes compared to all other HLA alleles (twofold increase) (P = 0.0035). We further found an inverse linear relationship between the number of Gag epitopes recognized and the plasma viral load (R = -0.36; P = 0.0016). Particularly, recognition of multiple epitopes within two regions of Gag (amino acids [aa] 1 to 75 and aa 248 to 500) was associated with the maintenance of a low steady-state viremia, even years after acute infection.     

Comparisons of CD8+ T Cells Specific for Human Immunodeficiency Virus,Hepatitis C Virus,and Cytomegalovirus Reveal Differences in Frequency,Immunodominance, Phenotype,and Interleukin-2 Responsiveness

To better understand the components of an effective immune response to human immunodeficiency virus (HIV), the CD8⁺ T-cell responses to HIV, hepatitis C virus (HCV), and cytomegalovirus (CMV) were compared with regard to frequency, immunodominance, phenotype, and interleukin-2 (IL-2) responsiveness. Responses were examined in rare patients exhibiting durable immune-mediated control over HIV, termed long-term nonprogressors (LTNP) or elite controllers, and patients with progressive HIV infection (progressors). The magnitude of the virus-specific CD8⁺ T-cell response targeting HIV, CMV, and HCV was not significantly different between LTNP and progressors, even though their capacity to proliferate to HIV antigens was preserved only in LTNP. In contrast to HIV-specific CD8⁺ T-cell responses of LTNP, HLA B5701-restricted responses within CMV pp65 were rare and did not dominate the total CMV-specific response. Virus-specific CD8⁺ T cells were predominantly CD27⁺45RO⁺ for HIV and CD27⁻45RA⁺ for CMV; however, these phenotypes were highly variable and heavily influenced by the degree of viremia. Although IL-2 induced significant expansions of CMV-specific CD8⁺ T cells in LTNP and progressors by increasing both the numbers of cells entering the proliferating pool and the number of divisions, the proliferative capacity of a significant proportion of HIV-specific CD8⁺ T cells was not restored with exogenous IL-2. These results suggest that immunodominance by HLA B5701-restricted cells is specific to HIV infection in LTNP and is not a feature of responses to other chronic viral infections. They also suggest that poor responsiveness to IL-2 is a property of HIV-specific CD8⁺ T cells of progressors that is not shared with responses to other viruses over which immunologic control is maintained.Gaining a better understanding of the immunologic control of human immunodeficiency virus type 1 (HIV-1) is among the most critical goals for the rational design of HIV vaccines and immunotherapies. Although most HIV-infected patients develop high-level viremia, CD4⁺ T-cell depletion, and progressive disease, a rare subgroup of patients variably termed long-term nonprogressors (LTNP) or elite controllers restrict HIV replication to below 50 copies of HIV RNA/ml plasma and remain disease free for up to 25 years without antiretroviral therapy (ART). Measurements of HIV-specific immune responses in these patients, in comparison with progressors, are providing insights into mechanisms that mediate immunologic control or loss of control in humans. Although the mechanisms of restriction of HIV replication remain incompletely understood, a number of lines of evidence suggest that it is mediated by HIV-specific CD8⁺ T cells (reviewed in reference 51). High frequencies of HIV-specific CD8⁺ T cells specific for the autologous virus are observed in both LTNP and untreated progressors, suggesting that differences in immunologic control are mediated not by quantitative but more likely by qualitative features of the immune response.A number of qualitative features of the HIV-specific CD8⁺ T-cell response of LTNP or progressors have recently been proposed as the cause of immunologic control or loss of control, respectively. HLA B*5701 is highly overrepresented in LTNP, and the HIV-specific CD8⁺ T-cell response is highly focused on B5701-restricted peptides in B*5701⁺ LTNP but not in B*5701⁺ progressors (19, 50). In addition, there is a difference in surface markers between HIV- and cytomegalovirus (CMV)-specific CD8⁺ T cells thought to represent differences in maturation of the T-cell response (8). The CD8⁺ T cells of progressors are diminished in proliferative capacity and perforin upregulation in response to autologous HIV-infected CD4⁺ T cells (49). Recently, it has been proposed that this diminished proliferative capacity is due to a lack of paracrine or autocrine interleukin-2 (IL-2) production by HIV-specific CD4⁺ T cells or CD8⁺ T cells (41, 42, 75). Interpretation of proliferation studies is complicated by the fact that the effects of IL-2 were measured on the basis of 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) dye dilution of major histocompatibility complex (MHC) tetramer-positive cells. Because cell division over 6 days is an exponential function, IL-2 may induce small increases in the percentage of cells dividing or in the number of cell divisions that can result in large changes in the percent CFSE^lo cells, and yet the majority of antigen-specific cells may not proceed through the cell cycle. In addition, there are very limited data regarding whether the features of immunodominance, surface phenotype, and IL-2 responsiveness of HIV-specific CD8⁺ T cells extend to other chronic virus infections.In the present study, we examined these qualitative features within the response to HIV, CMV, or hepatitis C virus (HCV) across patient groups. We observed that the high degree of focus upon B5701-restricted peptides found in LTNP does not extend to the HCV- or CMV-specific responses. The phenotype of HIV- or CMV-specific CD8⁺ T cells was highly variable and heavily influenced by the degree of viremia. In addition, when both the number of divisions and the percentage of cells dividing were analyzed, proliferation of HIV-specific CD8⁺ T cells was refractory to IL-2 stimulation, unlike that of CMV-specific cells. These results offer important insights into qualitative features of the HIV-specific CD8⁺ T-cell response, whether they extend to responses to other viruses, and whether they are associated with the presence or absence of immunologic control.     

Inclusion of Vpr accessory gene in a plasmid vaccine cocktail markedly reduces Nef vaccine effectiveness in vivo resulting in CD4 cell loss and increased viral loads in rhesus macaques

We compared the immunogenicity of plasmid vaccines containing multiple human immunodeficiency virus (HIV) antigens and found that covaccination with plasmids expressing HIV-1 14 kDa vpr gene product profoundly reduces antigen-specific CD8-mediated cytotoxic T-cell activity (CTL). Interestingly, Th1 type responses against codelivered antigens (pGag-Pol, pNef, etc.) encoded by the plasmid vaccines were suppressed. This suggested that vpr might compromise CD8 T-cell immunity in vivo during infection. A pilot primate vaccine study was designed to test the hypothesis to compare the following groups: unvaccinated controls, animals vaccinated without simean immunodeficiency virus (SIV)-Nef antigen plasmid, and animals covaccinated with the identical plasmid antigen and a plasmid construct encoding SIV Vpr/Vpx. Animals were subsequently challenged intrarectally with pathogenic SIVmac251 after the final vaccination of a multiple immunization protocol. Control animals were all infected and exhibited high viral loads and rapid CD4+ T-cell loss. In contrast, the Nef plasmid-vaccinated animals were also infected but exhibited preservation of CD4+ T-cells and a multilog reduction in viral load compared with controls. Animals covaccinated multiple times with the Nef vaccine and pVpr/Vpx plasmid suffered rapid and profound loss of CD4+ T-cells. These results have important implications for the design of multicomponent and particle vaccines for HIV-1 as well as for our understanding of HIV/SIV pathogenesis in vivo.     

Co-administration of plasmid-encoded granulocyte-macrophage colony-stimulating factor increases human immunodeficiency virus-1 DNA vaccine-induced polyfunctional CD4+ T-cell responses

T-cell based vaccines against human immunodeficiency virus (HIV) generate specific responses that may limit both transmission and disease progression by controlling viral load. Broad, polyfunctional, and cytotoxic CD4⁺T-cell responses have been associated with control of simian immunodeficiency virus/HIV-1 replication, supporting the inclusion of CD4⁺ T-cell epitopes in vaccine formulations. Plasmid-encoded granulocyte-macrophage colony-stimulating factor (pGM-CSF) co-administration has been shown to induce potent CD4⁺ T-cell responses and to promote accelerated priming and increased migration of antigen-specific CD4⁺ T-cells. However, no study has shown whether co-immunisation with pGM-CSF enhances the number of vaccine-induced polyfunctional CD4⁺ T-cells. Our group has previously developed a DNA vaccine encoding conserved, multiple human leukocyte antigen (HLA)-DR binding HIV-1 subtype B peptides, which elicited broad, polyfunctional and long-lived CD4⁺ T-cell responses. Here, we show that pGM-CSF co-immunisation improved both magnitude and quality of vaccine-induced T-cell responses, particularly by increasing proliferating CD4⁺ T-cells that produce simultaneously interferon-γ, tumour necrosis factor-α and interleukin-2. Thus, we believe that the use of pGM-CSF may be helpful for vaccine strategies focused on the activation of anti-HIV CD4⁺ T-cell immunity.     

HLA B*5701-positive long-term nonprogressors/elite controllers are not distinguished from progressors by the clonal composition of HIV-specific CD8+ T cells

To better understand the qualitative features of effective human immunodeficiency virus (HIV)-specific immunity, we examined the TCR clonal composition of CD8(+) T cells recognizing conserved HIV p24-derived epitopes in HLA-B*5701-positive long-term nonprogressors/elite controllers (LTNP/EC) and HLA-matched progressors. Both groups displayed oligoclonal HLA-B5701-restricted p24-specific CD8(+) T-cell responses with similar levels of diversity and few public clonotypes. Thus, HIV-specific CD8(+) T-cell responses in LTNP/EC are not differentiated from those of progressors on the basis of clonal diversity or TCR sharing.     

HLA class I-restricted T-cell responses may contribute to the control of human immunodeficiency virus infection, but such responses are not always necessary for long-term virus control

A rare subset of human immunodeficiency virus (HIV)-infected individuals maintains undetectable HIV RNA levels without therapy ("elite controllers"). To clarify the role of T-cell responses in mediating virus control, we compared HLA class I polymorphisms and HIV-specific T-cell responses among a large cohort of elite controllers (HIV-RNA < 75 copies/ml), "viremic" controllers (low-level viremia without therapy), "noncontrollers" (high-level viremia), and "antiretroviral therapy suppressed" individuals (undetectable HIV-RNA levels on antiretroviral therapy). The proportion of CD4(+) and CD8(+) T cells that produce gamma interferon (IFN-gamma) and interleukin-2 (IL-2) in response to Gag and Pol peptides was highest in the elite and viremic controllers (P < 0.0001). Forty percent of the elite controllers were HLA-B*57 compared to twenty-three percent of viremic controllers and nine percent of noncontrollers (P < 0.001). Other HLA class I alleles more common in elite controllers included HLA-B*13, HLA-B*58, and HLA-B*81 (P < 0.05 for each). Within elite and viremic controller groups, those with protective class I alleles had higher frequencies of Gag-specific CD8(+) T cells than those without these alleles (P = 0.01). Noncontrollers, with or without protective alleles, had low-level CD8(+) responses. Thus, certain HLA class I alleles are enriched in HIV controllers and are associated with strong Gag-specific CD8(+)IFN-gamma(+)IL-2(+) T cells. However, the absence of evidence of T cell-mediated control in many controllers suggests the presence of alternative mechanisms for viral control in these individuals. Defining mechanisms for virus control in "non-T-cell controllers" might lead to insights into preventing HIV transmission or preventing virus replication.     

Changes in paracrine interleukin-2 requirement, CCR7 expression, frequency, and cytokine secretion of human immunodeficiency virus-specific CD4+ T cells are a consequence of antigen load

Virus-specific CD4+ T-cell responses are thought to be required for the induction and maintenance of many effective CD8+ T-cell and B-cell immune responses in experimental animals and humans. Although the presence of human immunodeficiency virus (HIV)-specific CD4+ T cells has been documented in patients at all stages of HIV infection, many fundamental questions regarding their frequency and function remain. A 10-color, 12-parameter flow cytometric panel was utilized to examine the frequency, memory phenotype (CD27, CCR7, and CD45RA), and cytokine production (interleukin-2 [IL-2], gamma interferon, and tumor necrosis factor alpha) of CD4+ T cells specific for HIV antigens as well as for adenovirus, Epstein-Barr virus (EBV), influenza H1N1 virus, influenza H3N2 virus, cytomegalovirus, varicella-zoster virus (VZV), and tetanus toxoid in normal controls, long-term nonprogressors (LTNP), and HIV-infected patients with progressive disease on or off therapy. The HIV-specific CD4+ T-cell responses in LTNP and patients on therapy were similar in frequency, phenotype, and cytokine production to responses directed against adenovirus, EBV, influenza virus, and VZV. HIV-specific CD4+ T cells from patients off antiretroviral therapy demonstrated a shift towards a CCR7(-) CD45RA(-) phenotype and a reduced percentage of IL-2-producing cells. The alterations in cytokine production during HIV viremia were found to be intrinsic to the HIV-specific CD4+ T cells and caused a requirement for IL-2 supplied exogenously for proliferation to occur. These observations suggest that many previously described changes in HIV-specific CD4+ T-cell function and phenotype are a consequence of high levels of antigen in viremic patients. In addition, defects in function and phenotype of HIV-specific CD4+ T cells are not readily discernible in the context of antiretroviral therapy but rather are similar to responses to other viruses.     

Absence of immunodominant anti-Gag p17 (SL9) responses among Gag CTL-positive, HIV-uninfected vaccine recipients expressing the HLA-A*0201 allele

According to a number of previous reports, control of HIV replication in humans appears to be linked to the presence of anti-HIV-1 Gag-specific CD8 responses. During the chronic phase of HIV-1 infection, up to 75% of the HIV-infected individuals who express the histocompatibility leukocyte Ag (HLA)-A*0201 recognize the Gag p17 SLYNTVATL (aa residues 77-85) epitope (SL9). However, the role of the anti-SL9 CD8 CTL in controlling HIV-1 infection remains controversial. In this study we determined whether the pattern of SL9 immunodominance in uninfected, HLA-A*0201 HIV vaccine recipients is similar to that seen in chronically HIV-infected subjects. The presence of anti-SL9 responses was determined using a panel of highly sensitive cellular immunoassays, including peptide:MHC tetramer binding, IFN-gamma ELISPOT, and cytokine flow cytometry. Thirteen HLA-A*0201 vaccinees with documented anti-Gag CD8 CTL reactivities were tested, and none had a detectable anti-SL9 response. These findings strongly suggest that the pattern of SL9 epitope immunodominance previously reported among chronically infected, HLA-A*0201-positive patients is not recapitulated in noninfected recipients of Gag-containing canarypox-based candidate vaccines and may be influenced by the relative immunogenicity of these constructs.     

Recognition of a defined region within p24 gag by CD8+ T cells during primary human immunodeficiency virus type 1 infection in individuals expressing protective HLA class I alleles

Human immunodeficiency virus type 1 (HIV-1)-specific immune responses during primary HIV-1 infection appear to play a critical role in determining the ultimate speed of disease progression, but little is known about the specificity of the initial HIV-1-specific CD8(+) T-cell responses in individuals expressing protective HLA class I alleles. Here we compared HIV-1-specific T-cell responses between subjects expressing the protective allele HLA-B27 or -B57 and subjects expressing nonprotective HLA alleles using a cohort of over 290 subjects identified during primary HIV-1 infection. CD8(+) T cells of individuals expressing HLA-B27 or -B57 targeted a defined region within HIV-1 p24 Gag (amino acids 240 to 272) early in infection, and responses against this region contributed over 35% to the total HIV-1-specific T-cell responses in these individuals. In contrast, this region was rarely recognized in individuals expressing HLA-B35, an HLA allele associated with rapid disease progression, or in subjects expressing neither HLA-B57/B27 nor HLA-B35 (P < 0.0001). The identification of this highly conserved region in p24 Gag targeted in primary infection specifically in individuals expressing HLA class I alleles associated with slower HIV-1 disease progression provides a rationale for vaccine design aimed at inducing responses to this region restricted by other, more common HLA class I alleles.     

A Vaccine Encoding Conserved Promiscuous HIV CD4 Epitopes Induces Broad T Cell Responses in Mice Transgenic to Multiple Common HLA Class II Molecules

Current HIV vaccine approaches are focused on immunogens encoding whole HIV antigenic proteins that mainly elicit cytotoxic CD8+ responses. Mounting evidence points toward a critical role for CD4+ T cells in the control of immunodeficiency virus replication, probably due to cognate help. Vaccine-induced CD4+ T cell responses might, therefore, have a protective effect in HIV replication. In addition, successful vaccines may have to elicit responses to multiple epitopes in a high proportion of vaccinees, to match the highly variable circulating strains of HIV. Using rational vaccine design, we developed a DNA vaccine encoding 18 algorithm-selected conserved, “promiscuous” (multiple HLA-DR-binding) B-subtype HIV CD4 epitopes - previously found to be frequently recognized by HIV-infected patients. We assessed the ability of the vaccine to induce broad T cell responses in the context of multiple HLA class II molecules using different strains of HLA class II- transgenic mice (-DR2, -DR4, -DQ6 and -DQ8). Mice displayed CD4+ and CD8+ T cell responses of significant breadth and magnitude, and 16 out of the 18 encoded epitopes were recognized. By virtue of inducing broad responses against conserved CD4+ T cell epitopes that can be recognized in the context of widely diverse, common HLA class II alleles, this vaccine concept may cope both with HIV genetic variability and increased population coverage. The vaccine may thus be a source of cognate help for HIV-specific CD8+ T cells elicited by conventional immunogens, in a wide proportion of vaccinees.     

Cytotoxic T-lymphocyte (CTL) responses directed against regulatory and accessory proteins in HIV-1 infection

The HIV-1 regulatory proteins Tat and Rev and the accessory proteins Vpr, Vpu, and Vif are essential for viral replication, and their cytoplasmic production suggests that they should be processed for recognition by cytotoxic T lymphocytes. However, only limited data is available evaluating to which extent these proteins are targeted in natural infection and optimal cytotoxic T lymphocyte (CTL) epitopes within these proteins have not been defined. In this study, CTL responses against HIV-1 Tat, Rev, Vpr, Vpu, and Vif were analyzed in 70 HIV-1 infected individuals and 10 HIV-1 negative controls using overlapping peptides spanning the entire proteins. Peptide-specific interferon-gamma (IFN-gamma) production was measured by Elispot assay and flow-based intracellular cytokine quantification. HLA class I restriction and cytotoxic activity were confirmed after isolation of peptide-specific CD8+ T-cell lines. All regulatory and accessory proteins served as targets for HIV-1- specific CTL and multiple CTL epitopes were identified in functionally important regions of these proteins. In certain individuals HIV-1-specific CD8+ T-cell responses to these accessory and regulatory proteins contributed up to a third to the magnitude of the total HIV-1-specific CTL response. These data indicate that despite the small size of these proteins regulatory and accessory proteins are targeted by CTL in natural HIV-1 infection, and contribute importantly to the total HIV-1-specific CD8+ T-cell responses. These findings are relevant for the evaluation of the specificity and breadth of immune responses during acute and chronic#10; infection, and will be useful for the design and testing of candidate human immunodeficiency virus (HIV) vaccines.     

Predominant involvement of CD8+CD28- lymphocytes in human immunodeficiency virus-specific cytotoxic activity.

Distinct functional CD8+ T-cell populations have been observed during human immunodeficiency virus (HIV) infection. One of these functions is the inhibition of viral replication by a noncytotoxic mechanism, which was shown to be mediated by the CD8+CD28+ subpopulation. On the other hand, CD8+ T cells exert an HIV-specific cytotoxic activity. The present study shows that CD8+CD28- lymphocytes display this HIV-specific cytotoxic activity, which is detectable immediately after the cells are purified from peripheral blood. The CD28- population is also able to proliferate and to retain its cytotoxic activity after in vitro restimulation with autologous blast cells. Finally, HIV-specific cytotoxic T cells can be obtained in vitro from the CD8+CD28+ population.     

Consistent patterns in the development and immunodominance of human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T-cell responses following acute HIV-1 infection

Human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T-cell responses generated during acute infection play a critical role in the initial control of viremia. However, little is known about the viral T-cell epitopes targeted during acute infection or about their hierarchy in appearance and relative immunodominance over time. In this study, HIV-1-specific CD8+ T-cell responses in 18 acutely infected individuals expressing HLA-A3 and/or -B7 were characterized. Detailed analysis of CD8 responses in one such person who underwent treatment of acute infection followed by reexposure to HIV-1 through supervised treatment interruptions (STI) revealed recognition of only two cytotoxic T-lymphocyte (CTL) epitopes during symptomatic acute infection. HIV-1-specific CD8+ T-cell responses broadened significantly during subsequent exposure to the virus, ultimately targeting 27 distinct CTL epitopes, including 15 different CTL epitopes restricted by a single HLA class I allele (HLA-A3). The same few peptides were consistently targeted in an additional 17 persons expressing HLA-A3 and/or -B7 during acute infection. These studies demonstrate a consistent pattern in the development of epitope-specific responses restricted by a single HLA allele during acute HIV-1 infection, as well as persistence of the initial pattern of immunodominance during subsequent STI. In addition, they demonstrate that HIV-1-specific CD8+ T-cell responses can ultimately target a previously unexpected and unprecedented number of epitopes in a single infected individual, even though these are not detectable during the initial exposure to virus. These studies have important implications for vaccine design and evaluation.     

Characterization of human immunodeficiency virus type 1 (HIV-1) Gag- and Gag peptide-specific CD4(+) T-cell clones from an HIV-1-seronegative donor following in vitro immunization

Substantial evidence argues that human immunodeficiency virus type 1 (HIV-1)-specific CD4(+) T cells play an important role in the control of HIV-1 replication in infected individuals. Moreover, it is increasingly clear that an HIV vaccine should elicit potent cytotoxic lymphocyte and antibody responses that will likely require an efficient CD4(+) T-cell response. Therefore, understanding and characterizing HIV-specific CD4(+) T-cell responses is an important aim. Here we describe the generation of HIV-1 Gag- and Gag peptide-specific CD4(+) T-cell clones from an HIV-1-seronegative donor by in vitro immunization with HIV-1 Gag peptides. The Gag peptides were able to induce a strong CD4(+) T-cell immune response in peripheral blood mononuclear cells from the HIV-1-seronegative donor. Six Gag peptide-specific CD4(+) T-cell clones were isolated and their epitopes were mapped. The region of p24 between amino acids 201 and 300 of Gag was defined as the immunodominant region of Gag. A new T helper epitope in the p6 protein of Gag was identified. Two clones were shown to recognize Gag peptides and processed Gag protein, while the other four clones reacted only to Gag peptides under the experimental conditions used. Functional analysis of the clones indicated that both Th1 and Th2 types of CD4(+) T cells were obtained. One clone showed direct antigen-specific cytotoxic activity. These clones represent a valuable tool for understanding the cellular immune response to HIV-1, and the study provides new insights into the HIV-1-specific CD4(+) T-cell response and the induction of an anti-Gag and -Gag peptide cellular primary immune response in vitro.     

Putative immunodominant human immunodeficiency virus-specific CD8(+) T-cell responses cannot be predicted by major histocompatibility complex class I haplotype

Recent studies of human immunodeficiency virus (HIV)-specific CD8(+) T cells have focused on responses to single, usually HLA-A2-restricted epitopes as surrogate measures of the overall response to HIV. However, the assumption that a response to one epitope is representative of the total response is unconfirmed. Here we assess epitope immunodominance and HIV-specific CD8(+) T-cell response complexity using cytokine flow cytometry to examine CD8(+) T-cell responses in 11 HLA-A2(+) HIV(+) individuals. Initial studies demonstrated that only 4 of 11 patients recognized the putative immunodominant HLA-A2-restricted p17 epitope SLYNTVATL, suggesting that the remaining subjects might lack significant HIV-specific CD8(+) T-cell responses. However, five of six SLYNTVATL nonresponders recognized other HIV epitopes, and two of four SLYNTVATL responders had greater responses to HIV peptides restricted by other class I alleles. In several individuals, no HLA-A2-restricted epitopes were recognized, but CD8(+) T-cell responses were detected to epitopes restricted by other HLA class I alleles. These data indicate that an individual's overall CD8(+) T-cell response to HIV is not adequately represented by the response to a single epitope and that individual major histocompatibility complex class I alleles do not predict an immunodominant response restricted by that allele. Accurate quantification of total HIV-specific CD8(+) T-cell responses will require assessment of the response to all possible epitopes.     

Novel Mucosal DNA-MVA HIV Vaccination in Which DNA-IL-12 Plus Cholera Toxin B Subunit (CTB) Cooperates to Enhance Cellular Systemic and Mucosal Genital Tract Immunity

Induction of local antiviral immune responses at the mucosal portal surfaces where HIV-1 and other viral pathogens are usually first encountered remains a primary goal for most vaccines against mucosally acquired viral infections. Exploring mucosal immunization regimes in order to find optimal vector combinations and also appropriate mucosal adjuvants in the HIV vaccine development is decisive. In this study we analyzed the interaction of DNA-IL-12 and cholera toxin B subunit (CTB) after their mucosal administration in DNA prime/MVA boost intranasal regimes, defining the cooperation of both adjuvants to enhance immune responses against the HIV-1 Env antigen. Our results demonstrated that nasal mucosal DNA/MVA immunization schemes can be effectively improved by the co-delivery of DNA-IL-12 plus CTB inducing elevated HIV-specific CD8 responses in spleen and more importantly in genital tract and genito-rectal draining lymph nodes. Remarkably, these CTL responses were of superior quality showing higher avidity, polyfunctionality and a broader cytokine profile. After IL-12+CTB co-delivery, the cellular responses induced showed an enhanced breadth recognizing with higher efficiency Env peptides from different subtypes. Even more, an in vivo CTL cytolytic assay demonstrated the higher specific CD8 T-cell performance after the IL-12+CTB immunization showing in an indirect manner its potential protective capacity. Improvements observed were maintained during the memory phase where we found higher proportions of specific central memory and T memory stem-like cells T-cell subpopulations. Together, our data show that DNA-IL-12 plus CTB can be effectively employed acting as mucosal adjuvants during DNA prime/MVA boost intranasal vaccinations, enhancing magnitude and quality of HIV-specific systemic and mucosal immune responses.