Therapeutic Vaccination against the Rhesus Lymphocryptovirus EBNA-1 Homologue,rhEBNA-1, Elicits T Cell Responses to Novel Epitopes in Rhesus Macaques

Epstein-Barr virus (EBV) is a vaccine/immunotherapy target due to its association with several human malignancies. EBNA-1 is an EBV protein consistently expressed in all EBV-associated cancers. Herein, EBNA-1-specific T cell epitopes were evaluated after AdC–rhEBNA-1 immunizations in chronically lymphocryptovirus-infected rhesus macaques, an EBV infection model. Preexisting rhEBNA-1-specific responses were augmented in 4/12 animals, and new epitopes were recognized in 5/12 animals after vaccinations. This study demonstrated that EBNA-1-specific T cells can be expanded by vaccination.     

Distinct APC Subtypes Drive Spatially Segregated CD4+ and CD8+ T-Cell Effector Activity during Skin Infection with HSV-1

Efficient infection control requires potent T-cell responses at sites of pathogen replication. However, the regulation of T-cell effector function in situ remains poorly understood. Here, we show key differences in the regulation of effector activity between CD4⁺ and CD8⁺ T-cells during skin infection with HSV-1. IFN-γ-producing CD4⁺ T cells disseminated widely throughout the skin and draining lymph nodes (LN), clearly exceeding the epithelial distribution of infectious virus. By contrast, IFN-γ-producing CD8⁺ T cells were only found within the infected epidermal layer of the skin and associated hair follicles. Mechanistically, while various subsets of lymphoid- and skin-derived dendritic cells (DC) elicited IFN-γ production by CD4⁺ T cells, CD8⁺ T cells responded exclusively to infected epidermal cells directly presenting viral antigen. Notably, uninfected cross-presenting DCs from both skin and LNs failed to trigger IFN-γ production by CD8⁺ T-cells. Thus, we describe a previously unappreciated complexity in the regulation of CD4⁺ and CD8⁺ T-cell effector activity that is subset-specific, microanatomically distinct and involves largely non-overlapping types of antigen-presenting cells (APC).     

4-1BBL Enhances CD8+ T Cell Responses Induced by Vectored Vaccines in Mice but Fails to Improve Immunogenicity in Rhesus Macaques

T cells play a central role in the immune response to many of the world’s major infectious diseases. In this study we investigated the tumour necrosis factor receptor superfamily costimulatory molecule, 4-1BBL (CD137L, TNFSF9), for its ability to increase T cell immunogenicity induced by a variety of recombinant vectored vaccines. To efficiently test this hypothesis, we assessed a number of promoters and developed a stable bi-cistronic vector expressing both the antigen and adjuvant. Co-expression of 4-1BBL, together with our model antigen TIP, was shown to increase the frequency of murine antigen-specific IFN-γ secreting CD8⁺ T cells in three vector platforms examined. Enhancement of the response was not limited by co-expression with the antigen, as an increase in CD8⁺ immunogenicity was also observed by co-administration of two vectors each expressing only the antigen or adjuvant. However, when this regimen was tested in non-human primates using a clinical malaria vaccine candidate, no adjuvant effect of 4-1BBL was observed limiting its potential use as a single adjuvant for translation into a clinical vaccine.     

Human Immunodeficiency Virus Type 1-Specific CD8+ T-Cell Responses during Primary Infection Are Major Determinants of the Viral Set Point and Loss of CD4+ T Cells

Primary HIV-1 infection (PHI) is marked by a flu-like syndrome and high levels of viremia that decrease to a viral set point with the first emergence of virus-specific CD8⁺ T-cell responses. Here, we investigated in a large cohort of 527 subjects the immunodominance pattern of the first virus-specific cytotoxic T-lymphocyte (CTL) responses developed during PHI in comparison to CTL responses in chronic infection and demonstrated a distinct relationship between the early virus-specific CTL responses and the viral set point, as well as the slope of CD4⁺ T-cell decline. CTL responses during PHI followed clear hierarchical immunodominance patterns that were lost during the transition to chronic infection. Importantly, the immunodominance patterns of human immunodeficiency virus type 1 (HIV-1)-specific CTL responses detected in primary, but not in chronic, HIV-1 infection were significantly associated with the subsequent set point of viral replication. Moreover, the preservation of the initial CD8⁺ T-cell immunodominance patterns from the acute into the chronic phase of infection was significantly associated with slower CD4⁺ T-cell decline. Taken together, these data show that the specificity of the initial CTL response to HIV is critical for the subsequent control of viremia and have important implications for the rational selection of antigens for future HIV-1 vaccines.In the first weeks after human immunodeficiency virus type 1 (HIV-1) acquisition, viral loads peak at high levels, accompanied by a flu-like syndrome (15). A rapid depletion of the CD4⁺ T-cell population occurs during this acute infection, in particular, within the gastrointestinal tract-associated lymphoid tissue (6, 19, 20), marking a nonrecoverable scar on the immune system. With the resolution of the clinical syndromes, viral loads decrease to a set point, which persists at this level for months to years until progressive CD4⁺ T-cell decline results in the onset of AIDS. It has been shown that the initial viral set point following primary infection is a very strong predictor of the disease-free period until the onset of AIDS (18, 21, 22).The initial decrease in the viral load during primary HIV-1 infection (PHI) is temporally associated with the first emergence of virus-specific CD8⁺ T-cell responses, and several studies have provided strong evidence that HIV-1-specific CD8⁺ T-cell responses are capable of controlling viral replication (5, 16, 24, 25, 27, 31, 33). However, significant numbers of virus-specific CD8⁺ T cells are detectable both in chronically infected individuals who progress rapidly to AIDS and in those who do not experience HIV-1 disease progression for decades (1, 11), and the characteristics that define a protective HIV-1-specific CD8⁺ T-cell response are not known. In particular, the level of control over viral replication is not predicted by the overall breadth, magnitude, or function of virus-specific CD8⁺ T-cell responses in chronic HIV-1 infection (1, 4, 11, 26, 28).Here, we demonstrate in a large cohort of individuals identified during PHI that immunodominance patterns of virus-specific CD8⁺ T-cell responses detected in PHI, but not in chronic HIV-1 infection, are strongly associated with the subsequent set point of viral replication. These data show that the specificity of the initial CD8⁺ T-cell response to HIV is critical for the subsequent control of viremia and have important implications for the rational selection of antigens for future HIV-1 vaccines.     

Similar Impact of CD8+ T Cell Responses on Early Virus Dynamics during SIV Infections of Rhesus Macaques and Sooty Mangabeys

Despite comparable levels of virus replication, simian immunodeficiency viruses (SIV) infection is non-pathogenic in natural hosts, such as sooty mangabeys (SM), whereas it is pathogenic in non-natural hosts, such as rhesus macaques (RM). Comparative studies of pathogenic and non-pathogenic SIV infection can thus shed light on the role of specific factors in SIV pathogenesis. Here, we determine the impact of target-cell limitation, CD8+ T cells, and Natural Killer (NK) cells on virus replication in the early SIV infection. To this end, we fit previously published data of experimental SIV infections in SMs and RMs with mathematical models incorporating these factors and assess to what extent the inclusion of individual factors determines the quality of the fits. We find that for both rhesus macaques and sooty mangabeys, target-cell limitation alone cannot explain the control of early virus replication, whereas including CD8+ T cells into the models significantly improves the fits. By contrast, including NK cells does only significantly improve the fits in SMs. These findings have important implications for our understanding of SIV pathogenesis as they suggest that the level of early CD8+ T cell responses is not the key difference between pathogenic and non-pathogenic SIV infection.     

Stable Antigen Is Most Effective for Eliciting CD8+ T-Cell Responses after DNA Vaccination and Infection with Recombinant Vaccinia Virus In Vivo

The induction of strong CD8(+) T-cell responses against infectious diseases and cancer has remained a major challenge. Depending on the source of antigen and the infectious agent, priming of CD8(+) T cells requires direct and/or cross-presentation of antigenic peptides on major histocompatibility complex (MHC) class I molecules by professional antigen-presenting cells (APCs). However, both pathways show distinct preferences concerning antigen stability. Whereas direct presentation was shown to efficiently present peptides derived from rapidly degraded proteins, cross-presentation is dependent on long-lived antigen species. In this report, we analyzed the role of antigen stability on DNA vaccination and recombinant vaccinia virus (VV) infection using altered versions of the same antigen. The long-lived nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) can be targeted for degradation by N-terminal fusion to ubiquitin or, as we show here, to the ubiquitin-like modifier FAT10. Direct presentation by cells either transfected with NP-encoding plasmids or infected with recombinant VV in vitro was enhanced in the presence of short-lived antigens. In vivo, however, the highest induction of NP-specific CD8(+) T-cell responses was achieved in the presence of long-lived NP. Our experiments provide evidence that targeting antigens for proteasomal degradation does not improve the immunogenicity of DNA vaccines and recombinant VVs. Rather, it is the long-lived antigen that is superior for the efficient activation of MHC class I-restricted immune responses in vivo. Hence, our results suggest a dominant role for antigen cross-priming in DNA vaccination and recombinant VV infection.     

IL-4 Induced Innate CD8+ T Cells Control Persistent Viral Infection

Memory-like CD8⁺ T cells expressing eomesodermin are a subset of innate T cells initially identified in a number of genetically modified mice, and also exist in wild mice and human. The acquisition of memory phenotype and function by these T cells is dependent on IL–4 produced by PLZF⁺ innate T cells; however, their physiologic function is still not known. Here we found that these IL-4-induced innate CD8⁺ T cells are critical for accelerating the control of chronic virus infection. In CIITA-transgenic mice, which have a substantial population of IL-4-induced innate CD8⁺ T cells, this population facilitated rapid control of viremia and induction of functional anti-viral T-cell responses during infection with chronic form of lymphocytic choriomeningitis virus. Characteristically, anti-viral innate CD8⁺ T cells accumulated sufficiently during early phase of infection. They produced a robust amount of IFN-γ and TNF-α with enhanced expression of a degranulation marker. Furthermore, this finding was confirmed in wild-type mice. Taken together, the results from our study show that innate CD8⁺ T cells works as an early defense mechanism against chronic viral infection.     

The Selection of Low Envelope Glycoprotein Reactivity to Soluble CD4 and Cold during Simian-Human Immunodeficiency Virus Infection of Rhesus Macaques

Envelope glycoprotein (Env) reactivity (ER) describes the propensity of human immunodeficiency virus type 1 (HIV-1) Env to change conformation from the metastable unliganded state in response to the binding of ligands (antibodies and soluble CD4 [sCD4]) or incubation in the cold. To investigate Env properties that favor in vivo persistence, we inoculated rhesus macaques with three closely related CCR5-tropic simian-human immunodeficiency viruses (SHIVs) that differ in ER to cold (ER_cold) and ER to sCD4 (ER_sCD4); these SHIVs were neutralized by antibodies equivalently and thus were similar in ER_antibody. All three SHIVs achieved high levels of acute viremia in the monkeys without alteration of their Env sequences, indicating that neither ER_cold nor ER_sCD4 significantly influences the establishment of infection. Between 14 and 100 days following infection, viruses with high ER_cold and ER_sCD4 were counterselected. Remarkably, the virus variant with low ER_cold and low ER_sCD4 did not elicit a neutralizing antibody response against the infecting virus, despite the generation of high levels of anti-Env antibodies in the infected monkeys. All viruses that achieved persistent viremia escaped from any autologous neutralizing antibodies and exhibited low ER_cold and low ER_sCD4. One set of gp120 changes determined the decrease in ER_cold and ER_sCD4, and a different set of gp120 changes determined resistance to autologous neutralizing antibodies. Each set of changes contributed to a reduction in Env-mediated entry. During infection of monkeys, any Env replication fitness costs associated with decreases in ER_cold and ER_sCD4 may be offset by minimizing the elicitation of autologous neutralizing antibodies.     

Evolution of CD8+ T Cell Responses after Acute PARV4 Infection

PARV4 is a small DNA human virus that is strongly associated with hepatitis C virus (HCV) and HIV infections. The immunologic control of acute PARV4 infection has not been previously described. We define the acute onset of PARV4 infection and the characteristics of the acute-phase and memory immune responses to PARV4 in a group of HCV- and HIV-negative, active intravenous drug users. Ninety-eight individuals at risk of blood-borne infections were tested for PARV4 IgG. Gamma interferon enzyme-linked immunosorbent spot assays, intracellular cytokine staining, and a tetrameric HLA-A2–peptide complex were used to define the T cell populations responding to PARV4 peptides in those individuals who acquired infection during the study. Thirty-five individuals were found to be PARV4 seropositive at the end of the study, eight of whose baseline samples were found to be seronegative. Persistent and functional T cell responses were detected in the acute infection phase. These responses had an active, mature, and cytotoxic phenotype and were maintained several years after infection. Thus, PARV4 infection is common in individuals exposed to blood-borne infections, independent of their HCV or HIV status. Since PARV4 elicits strong, broad, and persistent T cell responses, understanding of the processes responsible may prove useful for future vaccine design.     

Novel Recombinant Mycobacterium bovis BCG,Ovine Atadenovirus,and Modified Vaccinia Virus Ankara Vaccines Combine To Induce Robust Human Immunodeficiency Virus-Specific CD4 and CD8 T-Cell Responses in Rhesus Macaques

Mycobacterium bovis bacillus Calmette-Guérin (BCG), which elicits a degree of protective immunity against tuberculosis, is the most widely used vaccine in the world. Due to its persistence and immunogenicity, BCG has been proposed as a vector for vaccines against other infections, including HIV-1. BCG has a very good safety record, although it can cause disseminated disease in immunocompromised individuals. Here, we constructed a recombinant BCG vector expressing HIV-1 clade A-derived immunogen HIVA using the recently described safer and more immunogenic BCG strain AERAS-401 as the parental mycobacterium. Using routine ex vivo T-cell assays, BCG.HIVA⁴⁰¹ as a stand-alone vaccine induced undetectable and weak CD8 T-cell responses in BALB/c mice and rhesus macaques, respectively. However, when BCG.HIVA⁴⁰¹ was used as a priming component in heterologous vaccination regimens together with recombinant modified vaccinia virus Ankara-vectored MVA.HIVA and ovine atadenovirus-vectored OAdV.HIVA vaccines, robust HIV-1-specific T-cell responses were elicited. These high-frequency T-cell responses were broadly directed and capable of proliferation in response to recall antigen. Furthermore, multiple antigen-specific T-cell clonotypes were efficiently recruited into the memory pool. These desirable features are thought to be associated with good control of HIV-1 infection. In addition, strong and persistent T-cell responses specific for the BCG-derived purified protein derivative (PPD) antigen were induced. This work is the first demonstration of immunogenicity for two novel vaccine vectors and the corresponding candidate HIV-1 vaccines BCG.HIVA⁴⁰¹ and OAdV.HIVA in nonhuman primates. These results strongly support their further exploration.Vaccine strategies must balance safety with immunogenicity. Recombinant attenuated subunit vaccines are generally regarded as safe, but not sufficiently immunogenic as stand-alone vaccines (17). Heterologous prime-boost regimens employing diverse attenuated viruses or bacteria as vectors delivering a common, often T cell-based, immunogen have been shown to induce stronger responses than multiple repeated dosings of the same vaccine modalities (19, 22, 39, 54). This is because heterologous regimens allow boosting of pathogen insert-specific responses while avoiding the accumulation of antivector immunity, which can significantly decrease vaccine “take” (1, 41). Results of the STEP study, which used a candidate single-vector human immunodeficiency virus type 1 (HIV-1) vaccine (6, 17, 41), have highlighted the need for novel alternative vaccine vectors and strategies. Such alternatives could complement the limited mainstream vectors and provide additional safety and immunogenicity through increased flexibility, for example, through the availability of personalized vaccination regimens based on preexisting immune status and/or responsiveness to vaccination.Mycobacterium bovis bacillus Calmette-Guérin (BCG) remains the world''s most widely used vaccine, with over three billion doses administered since its deployment in 1920s. It is the only licensed vaccine against tuberculosis and is administered at birth as part of the WHO Expanded Programme on Immunization (EPI). Due to its many attractive features, BCG or related mycobacterial vectors have also been explored in the context of vaccines against a number of infectious agents such as Leishmania, Borrelia burgdorferi, Streptococcus pneumoniae, Bordetella pertussis, malaria, cottontail rabbit papillomavirus, measles virus, and indeed human and simian immunodeficiency viruses (34). Many of these vaccines showed immunogenicity and protection in murine models, and some were also immunogenic in nonhuman primates (8, 56, 67, 68). In human adults, recombinant BCG (rBCG) vaccines alone failed to provide consistent protection against Lyme disease (13). In addition to adult applications, we have suggested the use of rBCG expressing an HIV-1-derived immunogen as the priming component of a vaccine platform against mother-to-child transmission of HIV-1 through infected breast milk (32), where it would be critical to elicit a protective HIV-1-specific response as soon as possible after birth.To compare vectors and heterologous prime-boost regimens directly, we have advocated and pioneered the development of a panel of vaccine modalities delivering the same shared immunogen (18). Our first such model immunogen is called HIVA (21). This is a T-cell immunogen comprising HIV-1 consensus clade A Gag and a string of partially overlapping immunodominant CD8 T-cell epitopes originating from Gag, Pol, Nef, and Env, which has already been tested extensively in human volunteers (20). To facilitate iterative preclinical improvements of the HIVA vaccines, epitopes recognized by murine (58) and rhesus macaque (44) CD8 T cells were also incorporated. Furthermore, we have formulated HIVA into various vaccine modalities, including plasmid DNA (21), modified vaccinia virus Ankara (MVA) (21), human adenovirus serotype 5 (HAdV-5) (5), Semliki Forest virus replicons (18, 49), recombinant lysine auxotroph BCG strain Pasteur (32), and baculovirus-expressed and purified, bluetongue virus-derived chimeric NS1 tubules (37); the immunogenicity of these vectors has been compared directly and in heterologous combinations. More recently, we reported on the immunogenicity of a novel and promising vaccine vector derived from ovine atadenovirus type 7 (OAdV) (5); OAdV is the prototype member of the genus Atadenovirus, which is structurally and biologically distinct from Mastadenovirus (e.g., HAdV-5) (2, 50). Importantly, no immunity to OAdV has so far been detected in human sera (26). In mice, OAdV.HIVA induced strong polyfunctional HIVA-specific T cell responses with distinct kinetics from those induced by HAdV5.HIVA and displayed demonstrable single-dose efficacy against a surrogate virus challenge (5). OAdV is approved for use in a phase I human clinical trial (http://clinicaltrials.gov identifier no. {"type":"clinical-trial","attrs":{"text":"NCT00625430","term_id":"NCT00625430"}}NCT00625430). All of the vectors/modalities we explore are perceived to be safe and acceptable for use in humans.Here, as a step toward translating our results into human volunteers, we constructed a novel vaccine designated BCG.HIVA⁴⁰¹ vectored by AERAS-401, a Danish 1331 strain of BCG with improved immunogenicity and safety (57), and demonstrated priming of T cells to the HIVA transgene product in rhesus macaques. These BCG.HIVA⁴⁰¹-primed HIV-1-specific CD4 and CD8 T-cell responses were readily boosted with MVA.HIVA and OAdV.HIVA vaccines to elicit broad and robust HIV-1-specific T cell responses.     

Early Gag Immunodominance of the HIV-Specific T-Cell Response during Acute/Early Infection Is Associated with Higher CD8+ T-Cell Antiviral Activity and Correlates with Preservation of the CD4+ T-Cell Compartment

The important role of the CD8⁺ T-cell response on HIV control is well established. Moreover, the acute phase of infection represents a proper scenario to delineate the antiviral cellular functions that best correlate with control. Here, multiple functional aspects (specificity, ex vivo viral inhibitory activity [VIA] and polyfunctionality) of the HIV-specific CD8⁺ T-cell subset arising early after infection, and their association with disease progression markers, were examined. Blood samples from 44 subjects recruited within 6 months from infection (primary HIV infection [PHI] group), 16 chronically infected subjects, 11 elite controllers (EC), and 10 healthy donors were obtained. Results indicated that, although Nef dominated the anti-HIV response during acute/early infection, a higher proportion of early anti-Gag T cells correlated with delayed progression. Polyfunctional HIV-specific CD8⁺ T cells were detected at early time points but did not associate with virus control. Conversely, higher CD4⁺ T-cell set points were observed in PHI subjects with higher HIV-specific CD8⁺ T-cell VIA at baseline. Importantly, VIA levels correlated with the magnitude of the anti-Gag cellular response. The advantage of Gag-specific cells may result from their enhanced ability to mediate lysis of infected cells (evidenced by a higher capacity to degranulate and to mediate VIA) and to simultaneously produce IFN-γ. Finally, Gag immunodominance was associated with elevated plasma levels of interleukin 2 (IL-2) and macrophage inflammatory protein 1β (MIP-1β). All together, this study underscores the importance of CD8⁺ T-cell specificity in the improved control of disease progression, which was related to the capacity of Gag-specific cells to mediate both lytic and nonlytic antiviral mechanisms at early time points postinfection.     

Age-Related CD4+CD25+Foxp3+ Regulatory T-Cell Responses During Plasmodium berghei ANKA Infection in Mice Susceptible or Resistant to Cerebral Malaria

Different functions have been attributed to CD4⁺CD25⁺Foxp3⁺ regulatory T-cells (Tregs) during malaria infection. Herein, we describe the disparity in Treg response and pro- and anti-inflammatory cytokines during infection with Plasmodium berghei ANKA between young (3-week-old) and middle-aged (8-month-old) C57BL/6 mice. Young mice were susceptible to cerebral malaria (CM), while the middle-aged mice were resistant to CM and succumbed to hyperparasitemia and severe anemia. The levels of pro-inflammatory cytokines, such as TNF-α, in young CM-susceptible mice were markedly higher than in middle-aged CM-resistant mice. An increased absolute number of Tregs 3-5 days post-inoculation, co-occurring with elevated IL-10 levels, was observed in middle-aged CM-resistant mice but not in young CM-susceptible mice. Our findings suggest that Treg proliferation might be associated with the suppression of excessive pro-inflammatory Th1 response during early malaria infection, leading to resistance to CM in the middle-aged mice, possibly in an IL-10-dependent manner.     

Two Kinetic Patterns of Epitope-Specific CD8 T-Cell Responses following Murine Gammaherpesvirus 68 Infection

Murine gammaherpesvirus 68 (γHV68) provides an important experimental model for understanding mechanisms of immune control of the latent human gammaherpesviruses. Antiviral CD8 T cells play a key role throughout three separate phases of the infection: clearance of lytic virus, control of the latency amplification stage, and prevention of reactivation of latently infected cells. Previous analyses have shown that T-cell responses to two well-characterized epitopes derived from ORF6 and ORF61 progress with distinct kinetics. ORF6₄₈₇-specific cells predominate early in infection and then decline rapidly, whereas ORF61₅₂₄-specific cells continue to expand through early latency, due to sustained epitope expression. However, the paucity of identified epitopes to this virus has limited our understanding of the overall complexities of CD8 T-cell immune control throughout infection. Here we screened 1,383 predicted H-2^b-restricted peptides and identified 33 responses, of which 21 have not previously been reported. Kinetic analysis revealed a spectrum of T-cell responses based on the rapidity of their decline after the peak acute response that generally corresponded to the expression patterns of the two previously characterized epitopes. The slowly declining responses that were maintained during latency amplification proliferated more rapidly and underwent maturation of functional avidity over time. Furthermore, the kinetics of decline was accelerated following infection with a latency-null mutant virus. Overall, the data show that γHV68 infection elicits a highly heterogeneous CD8 T-cell response that segregates into two distinctive kinetic patterns controlled by differential epitope expression during the lytic and latency amplification stages of infection.Murine gammaherpesvirus 68 (γHV68) is a mouse pathogen closely related to the human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi''s sarcoma-associated herpesvirus (KSHV). Intranasal infection of mice with γHV68 leads to an acute infection in lung epithelial cells that is ultimately cleared and the concurrent establishment of latency in B cells, dendritic cells, and macrophages that undergoes amplification in the spleen and is maintained lifelong (11, 12). Even though γHV68 has the capacity to downregulate major histocompatibility complex class I (MHC-I) molecules (36), CD8 T cells specific for γHV68 are generated and have been shown to proliferate in response to cognate antigen, protect naive mice from γHV68 infection, lyse peptide-pulsed target cells in vivo and in vitro, and maintain the ability to produce antiviral cytokines (5, 6, 13, 27, 35). Until recently, knowledge of the antiviral CD8 T-cell repertoire in C57BL/6 mice was largely limited to two well-characterized epitopes derived from ORF6 and ORF61. T-cell responses to these epitopes have been shown to progress with distinct kinetics, with ORF6₄₈₇-specific cells predominating early in infection and ORF61₅₂₄-specific cells continuing to expand through early latency before declining and then persisting at higher levels late in infection (33). The difference in response kinetics correlates with the differential presentation of the epitopes, with the ORF6₄₈₇ epitope being expressed only during lytic infection and the ORF61₅₂₄ epitope being expressed both during lytic infection and during the latency amplification phase (22, 28). Additionally, the latency amplification phase is associated with the expansion of CD8 T cells with a Vβ4 T-cell receptor (TCR) component in several mouse strains (17), presumably due to a superantigen-like effect of the γHV68 M1 protein (4, 9).To better understand the breadth of the anti-γHV68 T-cell response, we used an enzyme-linked immunospot (ELISpot) approach to identify new epitopes. We identified a large number of epitopes derived from 26 proteins that drive the acute CD8 T-cell response to γHV68, which then narrowed over time, resulting in a limited antiviral response during latency. We did not observe inflation of any of the responses, as has been demonstrated for some murine cytomegalovirus (MCMV)-specific responses (20, 26). There was no evidence for functional exhaustion, as all detectable CD8 T-cell responses maintained functionality, but the responses declined in numbers over time. The decline in responses occurred over a broad kinetic range, which segregated into two general groups that correlated precisely with those previously described for ORF6 and ORF61. Thus, some responses declined rapidly after the acute phase of infection, while others declined more slowly.We examined two epitope-specific responses from each of the two patterns in detail over time for functional and phenotypic characteristics and found the responses to be highly heterogeneous, differing in TCR affinity, functional avidity, and proliferation rates. Importantly, slowly declining responses were not maintained as efficiently after infection with a latency-deficient virus, consistent with a role for epitope expression in driving the heterogeneous rate of decline in cell number after the acute infection. The data show that the response kinetics seen for the ORF6₄₈₇ and ORF61₅₂₄ responses are broadly applicable to multiple CD8 T-cell epitopes.     

Treatment with IL-7 Prevents the Decline of Circulating CD4+ T Cells during the Acute Phase of SIV Infection in Rhesus Macaques

Although treatment with interleukin-7 (IL-7) was shown to transiently expand the naïve and memory T-cell pools in patients with chronic HIV-1 infection receiving antiretroviral therapy (ART), it is uncertain whether a full immunologic reconstitution can be achieved. Moreover, the effects of IL-7 have never been evaluated during acute HIV-1 (or SIV) infection, a critical phase of the disease in which the most dramatic depletion of CD4⁺ T cells is believed to occur. In the present study, recombinant, fully glycosylated simian IL-7 (50 µg/kg, s.c., once weekly for 7 weeks) was administered to 6 rhesus macaques throughout the acute phase of infection with a pathogenic SIV strain (mac251); 6 animals were infected at the same time and served as untreated controls. Treatment with IL-7 did not cause clinically detectable side effects and, despite the absence of concomitant ART, did not induce significant increases in the levels of SIV replication except at the earliest time point tested (day 4 post-infection). Strikingly, animals treated with IL-7 were protected from the dramatic decline of circulating naïve and memory CD4⁺ T cells that occurred in untreated animals. Treatment with IL-7 induced only transient T-cell proliferation, but it was associated with sustained increase in the expression of the anti-apoptotic protein Bcl-2 on both CD4⁺ and CD8⁺ T cells, persistent expansion of all circulating CD8⁺ T-cell subsets, and development of earlier and stronger SIV Tat-specific T-cell responses. However, the beneficial effects of IL-7 were not sustained after treatment interruption. These data demonstrate that IL-7 administration is effective in protecting the CD4⁺ T-cell pool during the acute phase of SIV infection in macaques, providing a rationale for the clinical evaluation of this cytokine in patients with acute HIV-1 infection.