Viral burden and disease progression in rhesus monkeys infected with chimeric simian-human immunodeficiency viruses

To determine the role of viral burden in simian-human immunodeficiency virus (SHIV)-induced disease, cellular provirus and plasma viral RNA levels were measured after inoculation of rhesus monkeys with four different SHIVs. These SHIVs included SHIV-HXBc2 and SHIV-89.6, constructed with env, tat, rev, and vpu derived from either cell line-passaged or primary patient isolates of human immunodeficiency virus type 1; the viral quasispecies SHIV-89.6P derived after in vivo passage of SHIV-89.6; and a molecular clone, SHIV-KB9, derived from SHIV-89.6P. SHIV-HXBc2 and SHIV-89.6 are nonpathogenic in rhesus monkeys; SHIV-89.6P and SHIV-KB9 cause rapid CD4(+) T cell depletion and an immunodeficiency syndrome. Relative SHIV provirus levels were highest during primary infection in monkeys infected with SHIV-89.6P, the virus that caused the most rapid and dramatic CD4(+) T cell depletion. However, by 10 weeks postinoculation, provirus levels were similar in monkeys infected with the pathogenic and nonpathogenic chimeric viruses. The virus infections that resulted in the highest peak and chronic viral RNA levels were the pathogenic viruses SHIV-89.6P and SHIV-KB9. SHIV-89. 6P uniformly caused rapid and profound CD4(+) T cell depletion and immunodeficiency. Infection with the SHIV-KB9 resulted in very low CD4(+) T cell counts without seroconversion in some monkeys and a substantial but less profound CD4(+) T cell depletion and rapid seroconversion in others. Surprisingly, the level of plasma viremia did not differ between SHIV-KB9-infected animals exhibiting these contrasting outcomes, suggesting that host factors may play an important role in AIDS virus pathogenesis.     

Comparison of vaccine strategies using recombinant env-gag-pol MVA with or without an oligomeric Env protein boost in the SHIV rhesus macaque model

Rhesus macaques were immunized with a replication-deficient vaccinia virus (MVA) expressing human immunodeficiency virus type 1 89.6 envelope (env) and SIV gagpol (MVA/SHIV89.6) with or without a protein boost consisting of soluble 89.6 env (gp140). Immunization with MVA/SHIV89.6 alone elicited binding antibodies in all animals and neutralizing antibodies in 5 of 15 animals. Both types of antibodies were enhanced by protein boosting. In addition, CD8 cells exhibiting CM9 tetramer binding were detected in the subset of animals that were Mamu-A*01 positive. Animals were challenged intravenously with either SHIV-89.6 (Study 1) or the more pathogenic derivative SHIV-89.6P (Study 2). In Study 1, all control and vaccinated animals except one became infected. However, the levels of viremia were as follows: controls > rMVA alone > rMVA + protein. The differences were statistically significant between immunized and control groups but not between the two immunized groups. In Study 2, all animals became infected; however, the vaccinated group exhibited a 5-fold reduction in peak viremia and a 10-fold reduction in the postacute phase viremia in comparison to the controls. All of the controls required euthanasia by 10 months after challenge. A relationship between vaccine-induced antibody titers and reduction in virus burden was observed in both studies. Thus, immunization with MVA/SHIV89.6 alone or with a protein boost stimulated both arms of the immune system and resulted in significant control of viremia and delayed progression to disease after challenge with SHIV-89.6P.     

Studies on GM-CSF DNA as an adjuvant for neutralizing Ab elicited by a DNA/MVA immunodeficiency virus vaccine

Here, we use a vaccine consisting of DNA priming followed by MVA boosting in rhesus macaques to investigate the ability of GM-CSF DNA to serve as an adjuvant for the elicitation of neutralizing Ab against an HIV-1 Env. The trial used Gag, Pol, and Env sequences from SHIV-89.6 in the immunogens and a neutralization escape variant of SHIV-89.6, SHIV-89.6P, for challenge. Co-delivery of GM-CSF and vaccine DNAs enhanced the temporal appearance of neutralizing Ab and broadened the specificity of the neutralizing activity to include SHIV-89.6P. Two long-term SHIV-89.6 infections elicited neutralizing activity for SHIV-89.6 but not SHIV-89.6P. Studies on the avidity of the anti-Env antisera revealed that the GM-CSF-adjuvanted vaccine had elicited higher avidity Ab than the non-adjuvanted vaccine or the infection. The GM-CSF-adjuvanted group showed a trend towards better control of the challenge infection and had better control of re-emergent virus (P < 0.01) than the non-adjuvanted group.     

The effect of early versus delayed challenge after vaccination in controlling SHIV 89.6P infection

We sought to determine how effectively a CD8+ T cell inducing vaccine controls SHIV-89.6P infection in rhesus macaques at a range of challenge times post-vaccination. To this end, twenty eight Mamu-A*01+ rhesus macaques were given replication incompetent human serotype 5 adenovirus vector expressing SIVmac239 gag DNA and boosted 24 weeks later. Groups of 4 monkeys were then challenged with SHIV-89.6P at 1, 3, 6, 12, and 24 weeks after the boost. We compared the kinetics of viral load, CD4+ and virus-specific CD8+ T cells in these macaques. Measurements of CD8+ T cells taken before challenge show an exponential decay between 1 and 12 weeks following vaccination (p<0.0001). After week 12, no further decay was observed. Twenty of 24 vaccinated animals maintained more CD4+ T cells and kept their viral load at least one order of magnitude lower than the control animals throughout the chronic phase of the study. All 24 vaccinated animals survived the duration of the study. The viral and T cell kinetics over the first two weeks differed between the vaccinated groups, with more recent vaccination improving the early control of virus (p-value=0.027). The rates of virus specific CD8+ T cell expansion were greater in animals having higher viral loads at one week (r=0.45, p=0.029), suggesting that the kinetics of early viral load may have a role in virus specific CD8+ T cell generation, although these early differences did not lead to different clinical outcomes within the vaccinated animals.     

GM-CSF DNA: an adjuvant for higher avidity IgG, rectal IgA, and increased protection against the acute phase of a SHIV-89.6P challenge by a DNA/MVA immunodeficiency virus vaccine

Single intradermal or intramuscular inoculations of GM-CSF DNA with the DNA prime for a simian-human immunodeficiency virus (SHIV)-89.6 vaccine, which consists of DNA priming followed by modified vaccinia Ankara (MVA) boosting, increased protection of both the blood and intestines against the acute phase of an intrarectal SHIV-89.6P challenge. GM-CSF appeared to contribute to protection by enhancing two antibody responses: the avidity maturation of anti-Env IgG in blood (p=or<0.01) and the presence of long lasting anti-viral IgA in rectal secretions (p<0.01). The avidity of anti-Env IgG showed strong correlations with protection both pre and post challenge. Animals with the highest avidity anti-Env Ab had 1000-fold reductions in peak viremia over those with the lowest avidity anti-Env Ab. The enhanced IgA response was associated with the best protection, but did not achieve significance.     

Immunogenicity in pig-tailed macaques of poliovirus replicons expressing HIV-1 and SIV antigens and protection against SHIV-89.6P disease

In the search for an effective vaccine against the human immunodeficiency virus (HIV), novel ways to deliver viral antigens are being evaluated. One such approach is the use of nonreplicating viral vectors encoding HIV and/or SIV genes that are expressed after infection of host cells. Nonreplicating poliovirus vectors, termed replicons, that expressed HIV-1/HXB2 and SIVmac239 gag and various HIV-1 env genes from different clades were tested for immunogenicity and protective efficacy against intravenous challenge of pig-tailed macaques with SHIV-89.6P. To maximize both cellular and humoral immune responses, a prime-boost regimen was used. Initially, macaques were immunized four times over 35 weeks by either the intranasal and intrarectal or the intramuscular (im) route with mixtures of poliovirus replicons expressing HIV-1 gag and multiple env genes. Immunization with replicons alone induced both serum antibodies and lymphocyte proliferative responses. After boosting with purified Env protein, neutralizing antibodies to SHIV-89.6P were induced in four of five immunized animals. In a second experiment, four macaques were immunized im three times over 27 weeks with replicons expressing the SIVmac239 gag and HIV-1/HXB2 env genes. All immunized animals were then boosted twice with purified HIV-1-89.6 rgp140-Env and SIVmac239 p55-Gag proteins. Four control animals received only the two protein inoculations. Immunized and control animals were then challenged intravenously with the pathogenic SHIV-89.6P. After challenge the animals were monitored for virus isolation from peripheral blood mononuclear cells and plasma viremia and for changes in virus-specific antibody titers. Na?ve pig-tailed macaques experienced rapid loss of CD4(+) T cells and died between 38 and 62 weeks after infection. In contrast, macaques immunized with replicons and proteins rapidly cleared plasma virus and did not experience sustained loss of CD4(+) lymphocytes. Furthermore, two of the four macaques that were immunized only with purified proteins maintained high viral burdens and lost greater than 95% of their CD4(+) lymphocytes within 2 to 4 weeks after challenge. Thus, poliovirus replicons expressing HIV-1 and SIV antigens were immunogenic in pig-tailed macaques and appeared to enhance the protective effects observed after administration of purified proteins alone.     

Enhancing of anti-viral activity against HIV-1 by stimulation of CD8+ T cells with thymic peptides.

HIV-1 can be neutralized by soluble factors produced and secreted by activated CD8+ T cells. Production of such anti-viral CD8 factors (including chemokines) can be induced with IL-2 or phytohaemagglutinin (PHA). In addition to PHA or IL-2, we have co-stimulated CD8+ T cells with PHA/IL-2 and a mixture of thymic peptides (TP) of molecular weights below 10 kD. For the activation, CD8+ T cells were purified from peripheral blood mononuclear cells of HIV-1- individuals and any resultant anti-viral activity was monitored using an HIV-1 neutralization assay. Using HIV-1 isolates highly resistant to chemokine inhibition we detected significantly higher levels of HIV-1 neutralizing activity in CD8+ T cell culture supernatants which had been co-activated with TP. When the TP-induced anti-viral activity was monitored, neutralization of both non-syncytia-inducing (NSI) and syncytia-inducing (SI) patient isolates was enhanced by 38% (NSI, PHA +/- TP), 66% (SI, PHA +/- TP), 28% (NSI, IL-2 +/- TP), and 57% (SI, IL-2 +/- TP) compared with the anti-viral activity present in supernatants from CD8+ T cell cultures stimulated only with PHA or IL-2. Peptide sequence analysis of purified TP showed that the TP mixture predominantly contains peptides with homology to human histone and collagen sequences. Our data demonstrate that CD8+ T cells are additionally activated by a mixture of TP. In this way, the production of HIV-1 neutralizing CD8 factors can be enhanced.     

Kinetics of expansion of SIV Gag-specific CD8+ T lymphocytes following challenge of vaccinated macaques

The ability of memory T cells to mount a recall response plays a key role in the ability of vaccinated animals to contain viral challenge. In this study, we intensively monitored the expansion of SIV Gag-specific CD8+ T cells in peripheral blood and tissues of rhesus macaques vaccinated with the attenuated strain SIVmac239Delta3 and challenged with the pathogenic viruses SIVmac239 or SIVsmE660. Although all vaccinated animals were infected with challenge virus, peak levels of plasma viremia in vaccinees were decreased by 1.5 to 2 logs as compared with naive controls. Decreased levels of plasma viremia in vaccinated animals were evident as early as 7 days post-challenge, well before the expansion of SIV-specific CD8+ T cells. Expansion of SIV-specific CD8+ T cells was not observed in peripheral blood or tissues until at least 14 days after infection and did not occur in most animals until after the initial peak of viral replication. The observation that expansion of SIV-specific CD8+ T cells is delayed until 7 days or more after initial detection of viremia highlights fundamental limitations in the ability of lentivirus-specific CD8+ T cells to mediate protection against challenge.     

Influence of peak viral load on the extent of CD4+ T-cell depletion in simian HIV infection

Simian HIV (SHIV) infection of macaques with CXCR4 tropic viruses results in early and profound CD4 T-cell depletion in the first few weeks of infection. Analyzing data from a large study of vaccination and SHIV-89.6P challenge, we observe a strong correlation between peak viral load and the extent of CD4 T-cell depletion in acute infection, consistent with a simple kinetic model of viral infection of CD4 T cells. We have modeled the dynamics of the interaction of virus and CD4 T cells over time to investigate the rate of CD4 T-cell infection and death. This analysis indicates that up to 80% of CD4 T cells are infected at peak viremia and that the proportion of CD4 T cells destroyed is correlated with the peak viral load. The simple relation between viral load and CD4 T-cell depletion allows prediction of the level of viral control required to prevent CD4 T-cell depletion in acute SHIV infection. Whether such a simple relation also holds for HIV or simian immunodeficiency virus infections remains to be determined, particularly in the gut and other anatomic sites in which most early T-cell depletion occurs.     

The role of interleukin-10 in the generation of CD4+ and CD8+ memory T cells (expressing a CD44+, CD62L- phenotype) and their contribution to the regulation of immunoglobulin E antibody formation

BACKGROUND: Immunization of mice with low doses of protein antigens like keyhole limpet hemocyanin (KLH) results in high immunoglobulin (Ig) E Ab titers in the sera of those mice while the application of high doses leads to the production of only marginal amounts of IgE but high levels of IgG2a and IgG1 antibodies. The aim of these studies is to elucidate the role of interleukin-10 (IL-10) in the generation of memory T cells and their contribution to the production of IgE Ab. METHODS: Both IL-10-deficient mice and control mice were immunized repeatedly with KLH. Serum levels of KLH-specific Ab were measured. The frequencies of memory T cells were determined by flow cytometry and the role of CD4+ and CD8+ T cells was evaluated. RESULTS: IL-10-deficient mice show an augmented production of IgE in vivo. They exhibit enhanced ratios of CD4+:CD8+ memory T cells with a CD44+, CD62L- phenotype with a significantly raised generation of CD4+ memory T cells. On the other hand, the development of CD8+ memory T cells is reduced moderately in IL-10-deficient mice, which is an interesting fact since it has been shown that primed CD8+ T cells suppress IgE Ab production at least in vitro. The ratios of total CD4+:CD8+ T cells are augmented in IL-10-deficient mice compared to wild-type mice and in K01 mice compared to K100 mice in vivo. CONCLUSIONS: The elevated ratios of CD4+:CD8+ T cells indicate a higher capacity to provide B cell help, which results in a strongly elevated IgE response in IL-10-deficient mice. These altered ratios are furthermore interesting in view of the regulatory role of CD8+ T cells which provide a suppressive potential regarding IgE Ab production as shown in vitro. The capacity of IL-10 to suppress IgE Ab production by reduction of the CD4+:CD8+ memory T cell ratio opens new possibilities in the interference with allergic disorders.     

Relationship of in vivo and ex vivo levels of TH1 and TH2 cytokines with viremia in HAART patients with and without opportunistic infections

TH1/TH2 cytokines' imbalance is critical to HIV-1 progression and pathogenesis. Opportunistic infections-related cytokine perturbations in the setting of highly active antiretroviral therapy (HAART) are unclear. The objective of this cross-sectional study was to identify the relationship between TH1/TH2 cytokines and viremia in HAART patients with/without opportunistic infections. Sera from 17 HAART patients with and 43 without opportunistic infections, and 20 HIV-seronegative controls were used to measure the levels of IL-2, IFN-gamma, IL-4, and IL-10 proteins and mRNAs by ELISA and RNase protection assays, respectively. Ex vivo cytokine production by the CD4+/CD8+ T cells from four low and four high viremia patients randomly selected from non-opportunistic infection group was also evaluated. Serum IL-2 and IFN-gamma levels were lower (P < 0.05) in patients than controls; this reduction was more pronounced for IFN-gamma in non-opportunistic infection patients. IL-4 and IL-10 were higher in patients than controls; this elevation was more remarkable in patients with opportunistic infections. Serum TH1/TH2 cytokine levels correlated with viremia. In vitro cytokine production assays showed that CD4+ T cells from low viremia patients mainly produced IL-2 and IFN-gamma, CD8+ T cells from high viremia patients produced IL-4, and both subsets comparably produced IL-10 in patients with similar viremia. Positive correlations between sera/supernatant proteins and cellular mRNAs were also found statistically significant (P < 0.05). It was therefore concluded that in vivo TH1/TH2 cytokine levels in HAART patients and their ex vivo production by the CD4+/CD8+ T cells correlated with viremia and were also modulated by the presence of opportunistic infections in these patients.     

Standardized assessment of NAb responses elicited in rhesus monkeys immunized with single- or multi-clade HIV-1 envelope immunogens

The genetic diversity of HIV-1 envelope glycoproteins (Env) remains a major obstacle to the development of an antibody-based AIDS vaccine. The present studies examine the breadth and magnitude of neutralizing antibody (NAb) responses in rhesus monkeys after immunization with DNA prime-recombinant adenovirus (rAd) boost vaccines encoding either single or multiple genetically distant Env immunogens, and subsequently challenged with a pathogenic simian-human immunodeficiency virus (SHIV-89.6P). Using a standardized multi-tier panel of reference Env pseudoviruses for NAb assessment, we show that monkeys immunized with a mixture of Env immunogens (clades A, B, and C) exhibited a greater breadth of NAb activity against neutralization-sensitive Tier 1 viruses following both vaccination and challenge compared to monkeys immunized with a single Env immunogen (clade B or C). However, all groups of Env-vaccinated monkeys demonstrated only limited neutralizing activity against Tier 2 pseudoviruses, which are more characteristic of the neutralization sensitivity of circulating HIV-1. Notably, the development of a post-challenge NAb response against SHIV-89.6P was similar in monkeys receiving either clade B, clade C, or clade A+B+C Env immunogens, suggesting cross-clade priming of NAb responses. In addition, vaccines encoding Env immunogens heterologous to SHIV-89.6P primed for a rapid anamnestic NAb response following infection compared to vaccines lacking an Env component. These results show that DNA/rAd immunization with multiple diverse Env immunogens is a viable approach for enhancing the breadth of NAb responses against HIV-1, and suggest that Env immunogens can prime for anamnestic NAb responses against a heterologous challenge virus.     

Higher levels of IL-18 circulate during primary infection of monkeys with a pathogenic SHIV than with a nonpathogenic SHIV

We have monitored kinetics of peripheral blood Interleukin (IL)-18 level, viral RNA load, and CD4(+) T cell counts in cynomolgus and rhesus macaques following infections of various simian/human immunodeficiency viruses (SHIVs) causing differential pathogenicity. Infections of cynomolgus and rhesus macaques with pathogenic SHIVs-C2/1 and -89.6PD, respectively, induced high levels of plasma IL-18 (0.1-1 ng/ml) and enhanced apoptosis of peripheral blood T cells during primary viremia, along with a rapid decline of CD4(+) T cells and a high level of set point viral load after primary viremia (six of six cases). In contrast, infections of cynomolgus macaques with nonpathogenic SHIVs-TH09V3 and -MD14 did not cause such IL-18 elevation, showing no decline of CD4(+) T cells and no or low viral set point level following primary viremia (three of three cases). Thus, the elevation of circulating IL-18 level during primary viral infection can be a good indicator of an active pathogenic viral infection. However, the role of increased IL-18 remains to be elucidated and needs further investigation.     

Recombinant vector-induced HIV/SIV-specific CD4 T lymphocyte responses in rhesus monkeys

The recently reported modest success of the RV144 Thai trial vaccine regimen in preventing HIV-1 acquisition has focused interest on the potential contribution to that protection of vaccine-elicited CD4⁺ T cell responses. We evaluated the induction of virus-specific CD4⁺ T cell responses in rhesus monkeys using a series of diverse vaccine vectors. We assessed both the magnitudes and functional profiles of the antigen-specific CD4⁺ T cells by measuring cytokine production, memory differentiation, and the expression of mucosal homing molecules. We found that DNA prime/recombinant MVA boost immunizations induced particularly high-frequency virus-specific CD4⁺ T cell responses with polyfunctional repertoires, and these responses were partially preserved following SHIV-89.6P challenge. The majority of the vaccine-elicited CD4⁺ T cells were CD28⁺ memory T cells that expressed low levels of β7. Neither the magnitudes nor the functional profiles of the virus-specific CD4⁺ T cells generated by vaccination were associated with a preservation of CD4⁺ T cells or control of viral replication following SHIV-89.6P challenge. Interestingly, monkeys primed with recombinant Ad5 immunogens showed a dramatic expansion of both the magnitude and polyfunctionality of the vaccine-elicited CD4⁺ T cell responses following envelope protein boost. These results demonstrate that vaccine strategies that include recombinant MVA or recombinant Ad5 vectors can elicit robust CD4⁺ T cell responses.     

Identification of a CD4 T cell epitope in the pneumonia virus of mice glycoprotein and characterization of its role in protective immunity

Pneumonia virus of mice (PVM) causes bronchiolitis and pneumonia in mice. Infection is associated with high levels of viral replication in the lungs and results in the functional inactivation of pulmonary virus-specific CD8 T cells. Due to its similarity to severe human respiratory syncytial virus (RSV) infection, PVM infection in mice has been proposed as an alternative RSV model. Here, we have delineated the minimal requirements for protective T cell immunity in the PVM model. Immunization with a CD8 T cell epitope from the PVM phosphoprotein P, combined with the ovalbumin (OVA) CD4 T cell epitope, did not confer protective immunity against lethal PVM challenge, suggesting a possible role of cognate CD4 T cell immunity. To determine the role of PVM-specific CD4 T cell responses, we mapped a PVM CD4 T cell epitope in the glycoprotein G, using a panel of overlapping peptides. Although immunization with this epitope provided some protection, solid protective immunity was only observed after immunization with a combination of the PVM-specific CD4 and CD8 T cell epitopes. Analysis of post-challenge T cell responses in immunized mice indicated that G-specific pulmonary CD4 T cells displayed a mixed Th1/Th2 phenotype, which was characterized by the presence of both IL-5 and IFN-gamma secreting cells, in the absence of overt pathology.     

Maintenance of CD8+ T cells during acute viral infection of the central nervous system requires CD4+ T cells but not interleukin-2

The JHM strain of mouse hepatitis virus (JHMV) is rapidly cleared from the central nervous system (CNS) by CD8(+) T cells. In the absence of CD4(+) T cells, fewer CD8(+) T cells are found within the CNS in association with a coordinate increase in apoptotic lymphocytes. Previous data suggested that CD4(+) T cells may support CD8(+) T cells through secretion of interleukin-2 (IL-2). To determine the in vivo role of IL-2 during CNS infection, IL-2 signaling was inhibited via administration of a neutralizing IL-2-specific monoclonal antibody (mAb). In contrast to depletion of CD4(+) T cells, inhibition of IL-2 signaling did not influence CD8(+) T cell infiltration, effector cell function or survival within the CNS. These data suggest that the cellular immune response to acute neurotropic JHMV infection requires a distinct CD4(+) T cell component, but is independent of a requirement for IL-2 for induction, activation, recruitment, and/or maintenance of CD8(+) T cells within the CNS during acute infection.     

Disparate effects of acute and chronic infection with SIVmac239 or SHIV-89.6P on macaque plasmacytoid dendritic cells

Blood plasmacytoid dendritic cells (pDCs) contribute to both innate and adaptive immune responses by secreting high levels of IFN-alpha following acute bacterial and viral infections and indirectly by augmenting cell-mediated immunity. Cross-sectional studies have shown that the number of circulating pDCs in HIV patients, compared to that in uninfected individuals, is reduced. However, since the time of infection is usually unknown in HIV-infected patients, pDC-virus interactions that occur immediately after virus exposure are poorly understood. The current study investigated pDC dynamics during acute and chronic infections of macaques with either SIVmac239 or the pathogenic SIV-HIV chimera, SHIV-89.6P, as models for HIV infection. In three rhesus and three pig-tailed macaques infected intravenously with SIVmac239, the percentages of pDCs in blood declined 2- to 6-fold during the first 6 weeks after infection and remained depressed throughout the disease course. Surprisingly, no consistent, comparable decline in peripheral blood pDCs was observed in six macaques infected with SHIV-89.6P. In this latter group, percentages of pDCs did not correlate with CD4(+) T cells, but there was an inverse relationship with viral load. In addition, when compared to na?ve controls, the percentages of pDCs were reduced in spleens and peripheral lymph nodes of SIVmac239- but not SHIV-89.6P-infected animals that had progressed to AIDS. Proviral DNA was detected during the acute phase in pDCs isolated from macaques infected with either virus. These results imply that, even though macaque pDCs can be infected by both SIVmac239 and SHIV-89.6P, the subsequent effects on in vivo pathogenesis differ. The underlying mechanism(s) for these differences is unclear, but the selection of SIV or SHIV as a challenge virus might influence the outcome of some studies, such as those evaluating vaccines or the therapeutic efficacy of drugs.     

Antiretroviral therapy-induced dominant interleukin-2 HIV-1 Gag CD4+ T cell response: evidence of functional recovery of HIV-1-specific CD4+ T cells

Prolonged antiretroviral treatment (ART) significantly changes the cytokine secretion capacities of HIV-1-specific T cells. However, it is unclear whether these changes result from decreased viremia or they correspond to true functional recovery of viral-specific immune response. To study this issue, we analysed the quantitative and qualitative differences of HIV-1-specific and polyclonal CD4+ and CD8+ T cells between 26 naive and 52 treated individuals. HIV-1 Gag and staphylococcal enterotoxin B (SEB)-reactive T cells were determined by flowcytometric intracellular secretion of IFN-γ or/and ΙL-2. ART resulted in increase of single IL-2 and decrease of single IFN-γ-secreting HIV-1 CD4+ T cells, while both cytokines secreting HIV-1 CD4+ T cells were presented in comparable frequencies in both groups. Viral loads correlated negatively with single IL-2 and positively with single IFN-γ-secreting HIV-1 CD4+ cells. Single IL-2 HIV-1 CD4+ T cells correlated positively with both cytokines secreting polyclonal CD8+ T cells. By qualitative analysis, a dominant IL-2 HIV-1 CD4+ T cell response (> 70% single IL-2) was identified only in ART suppressed patients, who also generated increased dual specific polyclonal CD8+ T cells. Polyfunctional HIV-1 CD4+ T cell responses were detected even in naive individuals with high viremia. In conclusion, the presence of dominant IL-2 HIV-1 CD4+ T cell response, associated with increased CD8+ T cells capable to produce IL-2, indicates that the recovery of HIV-1-specific CD4+ T cell functionality under ART is a feasible goal. Furthermore, polyfunctional HIV-1 CD4+ T cell responses seem not to be directly involved in viral replication control.     

Vaccine-elicited immune responses prevent clinical AIDS in SHIV(89.6P)-infected rhesus monkeys

Accumulating evidence has demonstrated the importance of cytotoxic T lymphocytes (CTLs) and helper T lymphocytes in controlling HIV-1 replication. We have elicited immune responses in rhesus monkeys utilizing DNA vaccines augmented by the administration of IL-2/Ig, a fusion protein consisting of interleukin-2 and the Fc portion of IgG2. These vaccine-elicited immune responses did not prevent infection following a high-dose intravenous challenge with SHIV(89.6P) but did control viremia to nearly undetectable levels and prevented immunodeficiency and clinical disease. In contrast, control monkeys developed high levels of viremia and exhibited a rapid loss of CD4(+) T cells, significant clinical disease progression, and death in half of the animals by day 140 following challenge. Vaccine approaches that elicit immune responses capable of reducing plasma viral loads, but not capable of inducing sterilizing immunity, may still provide substantial clinical benefits.     

Characterization of simian and human immunodeficiency chimeric viruses re-isolated from vaccinated macaque monkeys after challenge infection

Monkeys that have been vaccinated with nef-deleted SHIVs were either fully or partially protected against challenge with acute pathogenic SHIV-89.6 P. Viruses isolated from these vaccinated monkeys were all found to be the 89.6 P challenge virus using PCR amplification and restriction enzyme analysis of the env region of the viruses. Analysis of the 3'-end of the env region and 5'-half of the nef region using a heteroduplex mobility assay revealed that the parental 89.6 P and re-isolated viruses from unvaccinated 89.6 P-infected monkeys had quite an abundant and similar heterogeneous quasispecies population. In contrast, the viruses isolated from the vaccinated monkeys had different and fewer quasispecies indicating a selective immune pressure in the vaccinated monkeys. The in vitro replication of the viruses isolated from the vaccinated monkeys in human and macaque peripheral blood mononucular cells (PBMCs) as well as in established cell lines such as M8166 and HSC-F cells, were slow and delayed when compared to the parental 89.6 P and re-isolated viruses from unvaccinated 89.6 P-infected monkeys. Further comparison revealed that in HSC-F cells the viruses from vaccinated monkeys again showed delayed and weak CD4(+) cell down-modulation as well as having little or no effect on cell growth or cell viability on HSC-F cells and monkey PBMC. Thus we noticed that these re-isolated 89.6 P viruses from the vaccinated monkeys had changed or had been selected for low pathogenic viruses in the monkeys. This suggests that though the vaccination did not completely prevent the replication of the challenge virus in the monkeys it did contain the challenge virus by suppressing the pathogenic variants. This further enhances the prospects of this nef-deleted SHIV as the bases for effective anti-HIV vaccine candidates.