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Early HIV-1 infection is marked by rapid evolution of both CD8(+) T lymphocyte (CTL) epitope targeting and viral sequences, while chronic infection demonstrates relative stability of these parameters. To examine the interactions of changing CTL targeting and viremia in early infection, we assessed CTL targeting and viremia levels in persons during early HIV-1 infection (estimated 15-271 days post-infection) who were placed on effective antiretroviral therapy. Pre-therapy, CTL targeting of viral proteins varied between persons depending on time after infection. Across individuals, increasing time after infection was associated with increasing Gag and Pol targeting, suggesting increasing targeting of conserved sequences. The intensity of Gag targeting correlated to lower viremia levels, while Env targeting correlated to higher viremia levels during early infection. This suggested that shifted targeting towards more conserved sequences is involved with the drop of viremia during early infection, consistent with prior observations of correlation between Gag targeting and lower viremia during chronic infection. After suppressive antiretroviral therapy, CTL targeting was generally static, indicating that HIV-1 replication and evolution drives the evolution of CTL targeting in early infection. Overall, these data suggest that early CTL targeting is directed towards more variable epitopes, causing escape and re-targeting until more conserved epitopes are recognized stably in chronic infection. Circumventing this natural history by pre-targeting CTL against more conserved epitopes with a vaccine could minimize the initial period of viral escape and immune damage during acute infection, improving long-term containment of HIV-1.

The presence of antibody to R7V (anti-R7VAb), a seven-amino acid sequence derived from beta(2)-microglobulin incorporated into HIV-1 virions from the surface of infected cells, has been proposed as an early marker of nonprogressive HIV-1 infection. The present study was undertaken because no prospective studies have tested this hypothesis. Stored samples collected prospectively from 361 HIV-1 seroconverting men in the Multicenter AIDS Cohort Study (0.44-1.53 years after seroconversion) were assayed for the presence or absence of anti-R7VAb, using a standardized ELISA. Using Cox proportional hazards models, crude and adjusted relative hazards (RH) were determined for the following outcomes: (a) clinically defined AIDS, (b) clinically defined AIDS or CD4 T cell count of200 cells/microl, and (c) death. A total of 143 (39.6%) men had early anti-R7VAb and 218 (60.4%) did not; 192 (53.2%) developed AIDS. At the visit tested, men with anti-R7VAb had significantly lower CD4 T cell counts and higher plasma HIV-1 viral loads than those without antibody. After adjustment for CD4 T cell count, HIV-1 viral load, CCR5 polymorphism, and use of combined antiretroviral therapy, the presence of anti-R7VAb was associated with a higher risk of progression for all outcomes, but not significantly so. Absence of anti-R7VAb was significantly associated with expression of HLA-B*5701 and -B*2705, two alleles associated with slower progression of HIV-1 disease. The early presence of anti-R7VAb in HIV-1 seroconverters was not associated with slower progression of HIV-1 disease.

To infect cells, HIV-1 must bind to both the CD4 protein and another transmembrane “coreceptor.” Early in the course of disease, HIV-1 variants usually bind to the chemokine coreceptor CCR5; such variants are termed “R5 viruses.” Viruses using another chemokine coreceptor, CXCR4, termed “X4 viruses,” emerge later in disease progression in ~50% of individuals infected with clade B virus [1], although a higher percentage has been suggested on the basis of studies that used sensitive detection techniques [2]. The presence of X4 viruses has been associated with accelerated disease progression and a decrease in CD4+ T cells [3, 4], but despite descriptions of in vitro cytopathicity of X4 viruses [5-7], no causal in vivo relationship or mechanism for this association has been proven [8, 9]. In 3 studies of coreceptor evolution during untreated HIV-1 infection [3, 4, 10], X4 viruses tended to emerge just before the beginning of the decrease in the total (CD3+) T cell count (i.e., the CD3+ inflection point) that presages the onset of an AIDS-defining illness [11]. The ability of HIV-1 to bind to CXCR4 may facilitate destruction of naive T cells, which preferentially express CXCR4 as opposed to CCR5 [8], and thus may contribute to more rapid depletion of T cells. The uncertainty surrounding mechanisms of selection and evolution of X4-using HIV-1 and the inability to predict the emergence of these strains have taken on greater importance since the development of CCR5 antagonists as inhibitors of HIV-1 replication [12,13]. For example, in a recent study of monotherapy with a CCR5 antagonist, X4 viruses present in low amounts replaced the R5 population shortly after commencement of therapy [14]. This has led to concern that use of CCR5 antagonists may predispose to selection of CXCR4-tropic viruses and, thus, to faster progression of HIV-1 disease than would occur without blockade of CCR5. To further characterize the emergence of X4 strains during HIV-1 infection, we undertook a systematic analysis of HIV-1 coreceptor tropism in men from the Multicenter AIDS Cohort Study (MACS; study Web site available at: http://www.statepi.jhsph.edu/macs/macs.html) who were selected for this study in order to reflect the full spectrum of rates of HIV-1 disease progression. Our specific goals were to evaluate (1) the timing of X4 virus emergence with respect to CD3+ T cell decline and development of an AIDS-defining event, (2) the degree to which emergence of X4 viruses correlates with an increased risk of rapid disease progression, and (3) whether determination of the CD4+ T cell count can be an accurate screen for detectable levels of CXCR4-tropic viruses.

Mucosal immune responses induced by HIV-1 vaccines are likely critical for prevention. We report a Phase 1 safety and immunogenicity trial in eight participants using the vaccinia-based TBC-3B vaccine given subcutaneously to determine the relationship between HIV-1 specific systemic and gastrointestinal mucosal responses. Across all subjects, detectable levels of blood vaccinia- and HIV-1-specific antibodies were elicited but none were seen mucosally. While the vaccinia component was immunogenic for CD8 + T lymphocyte (CTL) responses in both blood and mucosa, it was greater in blood. The HIV-1 component of the vaccine was poorly immunogenic in both blood and mucosa. Although only eight volunteers were studied intensively, the discordance between mucosal and blood responses may highlight mechanisms contributing to recent vaccine failures. © 2008 Elsevier Ltd. All rights reserved.

Human immunodeficiency virus type 1 (HIV-1) evolves in vivo under selective pressure from CD8 + T-lymphocyte (CTL) responses, which are in turn determined by host and viral genetic factors, such as restricting major histocompatibility complex molecules and the available viral epitope sequences. However, CTL are derived stochastically through the random gene rearrangements to produce T-cell receptors (TCR), and the relative impact of genetic versus stochastic processes on CTL targeting of HIV and immune-driven viral evolution is unclear. Here we evaluate identical twins infected with HIV-1 as neonates from a common blood transfusion, with subsequently similar environmental exposures, thereby allowing controlled comparisons of CTL targeting and viral evolution. Seventeen years after infection, their CTL targeting of HIV-1 was remarkably similar. In contrast, their overall TCR profiles were highly dissimilar, and a dominant epitope was recognized by distinctly different TCR in each twin. Furthermore, their viral epitopes had diverged, and there was ongoing viral phylogenetic divergence between the twins between 12 and 17 years after infection. These results indicate that while CTL targeting is predominately genetically determined, stochastic influences render the interaction of HIV-1 and host immunity, and therefore viral escape and CTL efficacy, unpredictable.

Live attenuated HIV vaccines offer a means to introduce exogenous sequences into the viral genome to target the virus elimination in vivo. Foreign genes inserted into the nef region of HIV-1 NL4-3 were found to be rapidly deleted following virus infection and/or replication, in a size dependent manner, in the human fetal Thymus/Liver implants of severe combined immunodeficient mouse (SCID-hu) model. When the murine heat stable antigen (HSA) of 283 bp was substituted into HIV-1 nef region, the viral loads in vivo were comparable to the negative control nef attenuated HIV-1, and the reporter HSA gene was not deleted upon infection. However, the murine Thy1.2 gene (505 bp) substituted into the nef attenuated HIV-1, upon infection and replication, deleted 441 bp in vitro and 437 bp in vivo, of the inserted Thy1.2 gene. When the enhanced green fluorescence protein (eGFP) gene (720 bp) was substituted for nef, virus replication was aborted in vivo in the Thy/Liv implants, as seen by the background levels of viral loads, comparable to mock infected implants, and the eGFP gene was deleted. When the herpes simplex virus thymidine kinase gene, HSV-TK (1.15 kbp), or HSA gene, was substituted into the viral vpr gene, TK but not HSA gene was deleted, upon infection in vitro. Moreover, NL-TKI reporter virus with both intact nef and vpr genes shows deletion of TK gene both in vitro and in vivo. Excision of foreign genes occurred within the exogenous segments but not in the viral own regions. These results suggest that larger "suicide" genes introduced via HIV-1 can be deleted upon infection. However, smaller size nucleotide sequences or genes (approximately 300 bp) inserted in place of viral nef or vpr gene may be used to target the virus or its components, for attack and elimination in vivo, and thus have implications for the development of live attenuated HIV vaccines.

Nef is an HIV-1 protein that is absent in most retroviruses, yet its reading frame is highly maintained despite frequent targeting by CD8+ CTL in vivo. Because Nef is not necessarily required for viral replication, this consistent maintenance suggests that Nef plays an important role(s) and substantial fitness constraints prevent its loss in vivo. The ability of Nef to down-regulate cell surface MHC class I (MHC-I) molecules and render infected cells resistant to CTL in general is likely to be an important contributing function. We demonstrate that mutational escape of HIV-1 from Nef-specific CTL in vitro leads to progeny virions that are increased in their susceptibility to CTL of specificities for proteins other than Nef. The escape mutants contain multiple nef mutations that impair the ability of the virus to down-regulate MHC-I through disruption of its reading frame as well as epitope point mutations. Given the rarity of nef frameshifts in vivo, these data support the concept that the ability to down-regulate MHC-I could be a key constraint for preservation of Nef in vivo.

The host immune factors that determine susceptibility to HIV-1 infection are poorly understood. We compared multiple immunologic parameters in three groups of HIV-1-seronegative men: 14 highly exposed (HR10), 7 previously reported possibly to have sustained transient infection (PTI), and a control group of 14 low risk blood bank donors (BB). Virus-specific cellular immune assays were performed for CD4(+) T helper cell responses, CD8(+) cytotoxic T lymphocyte activity, CD8(+) cell chemokine release, and CD8(+) cell-derived antiviral soluble factor activity. General immune parameters evaluated included CCR5 genotype and phenotype, interferon alpha production by PBMCs, leukocyte subset analysis, and detailed T lymphocyte phenotyping. Comparisons revealed no detectable group-specific differences in measures of virus-specific immunity. However, the HR10 group differed from the BB group in several general immune parameters, having higher absolute monocyte counts, higher absolute CD8(+) T cell counts and percentages, lower naive and higher terminal effector CD8(+) cells, and lower levels of CD28(+)CD8(+) cells. These changes were not associated with seropositivity for other chronic viral infections. The PTI men appeared to have normal levels of monocytes and slightly elevated levels of CD8(+) T cells (also with increased effector and decreased naive cells). Although we cannot entirely exclude the contribution of other chronic viral infections, these findings suggest that long-lived systemic cellular antiviral immunity as detected by our assays is not a common mechanism for resistance to infection, and that resistance may be multifactorial. General immune parameters reflected by CD8(+) T cell levels and activation, and monocyte concentrations may affect the risk of infection with HIV-1, and/or serve as markers of exposure.

Epidemiologic studies have identified cocaine as a cofactor for development of acquired immunodeficiency syndrome (AIDS). To evaluate this interaction, human peripheral blood leukocytes (PBL) were implanted into severe combined immunodeficient mice and infected with human immunodeficiency virus (HIV) in both the presence and absence of cocaine. Concurrent administration of cocaine resulted in significantly more PBL becoming infected with HIV in vivo (38.85% vs. 18.5%). The number of CD4(+) cells recovered from HIV-infected, cocaine-treated animals was significantly lower than that from mice infected with HIV in the absence of cocaine (6.5 x 10(4) vs. 19 x 10(4)) and was associated with a lower CD4:CD8 ratio and a dramatic increase in virus load. Exposure to cocaine alone did not affect the implantation of PBL, suggesting a specific interaction between cocaine and HIV. This report describes a model for evaluating HIV cofactors and supports cocaine's role in the development and progression of AIDS.

Human immunodeficiency virus (HIV)-infected individuals exhibit a variety of hematopoietic dysfunctions. The SCID-hu mouse (severe combined immunodeficient mouse transplanted with human fetal thymus and liver tissues) can be used to model the loss of human hematopoietic precursor cell function following HIV infection and has a distinct advantage in that data can be obtained in the absence of confounding factors often seen in infected humans. In this study, we establish that HIV type 1 (HIV-1) bearing a reporter gene inserted into the viral vpr gene is highly aggressive in depleting human myeloid and erythroid colony-forming precursor activity in vivo. Human CD34 + progenitor cells can be efficiently recovered from infected implants yet do not express the viral reporter gene, despite severe functional defects. Our results indicate that HIV-1 infection alone leads to hematopoietic inhibition in vivo; however, this effect is due to indirect mechanisms rather than to direct infection of CD34 + cells in vivo.

Our understanding of human immunodeficiency virus type 1 (HIV-1)-induced pathogenesis is hampered by the inability to detect HIV-1 gene expression in infected viable cells. In this report, we describe two HIV-1 reporter constructs that are replication competent and cytopathic in vivo. These constructs contain DNA regions of two different lengths that bear the cDNA for the murine heat-stable antigen in the vpr region of a CXCR4-tropic virus. We used the SCID-hu mouse model and these reporter viruses to perform detailed kinetic studies of HIV-1 infection of human thymocytes in vivo. We document that the CD4 + /CD8 + thymocytes are the first to express virus and that this subset demonstrates the most rapid and extensive HIV-1-induced cell depletion. Following depletion of this subset, subsequent virus expression occurs predominantly in phenotypically CD4 − cells, suggesting that CD4 down-regulation occurs in HIV-1-infected thymocytes in vivo. These results demonstrate the utility of these HIV-1 reporter constructs to monitor HIV pathogenesis in vitro and in vivo.

Late-stage HIV-1 disease in humans has been associated with perturbations of the T cell receptor (TCR) Vbeta repertoire. It is not known if the observed loss of certain Vbeta families is attributable directly to HIV-1 infection or whether this is a consequence of multiple opportunistic infections. Putative HIV-1-associated superantigens have been postulated to be the cause of the perturbed TCR Vbeta repertoire and the subsequent CD4+ T cell depletion in HIV-1-infected humans. In this study, we examined the human TCR Vbeta repertoire in SCID-hu mice, housed in a pathogen-free environment and infected with a molecularly cloned virus strain, to ascertain directly the effect of HIV-1 on the human TCR Vbeta repertoire in the absence of other infectious agents. We demonstrate that mock-infected human thymus/liver (Thy/Liv) implants in SCID-hu mice have complete TCR Vbeta repertoires, reflective of a normal human thymus. However, HIV-1-infected implants in SCID-hu mice had depleted TCR Vbeta repertoires, corresponding with thymocyte depletion. These results indicate that HIV-1-specific mechanisms are the cause of the TCR Vbeta repertoire depletion in infected implants. However, these thymocyte depletions were not restricted to specific TCR Vbeta subsets. These results are not consistent with the hypothesis that HIV-1 acts as a superantigen in vivo. The disruption of the TCR Vbeta repertoire in the human Thy/Liv implants of the SCID-hu mice suggests that HIV-1 infection may be influencing T cell development in the thymus, contributing to both the overall CD4+ T cell depletion in AIDS and limited TCR repertoire diversity.

Recent studies suggest that HIV-specific antibody-dependent cell-mediated cytotoxicity (ADCC) antibodies contribute to protective immunity against HIV. An important characteristic of future HIV vaccines will, therefore, be the ability to stimulate production of these antibodies in both men and women. Early studies suggest that men may have a better ADCC antibody response against HIV than women. Our objective was to determine whether men and women differ with respect to their ADCC response to HIV-1 gp120. HIV-positive, asymptomatic untreated men and women were matched for race, age, CD4(+) T cell number, HIV-1 viral load, and treatment and HIV-1 gp120 ADCC antibody titers were compared. A standard (51)Cr-release assay was used to determine HIV-1 gp120 ADCC antibody titers in HIV-1-seropositive individuals from the Multicenter AIDS Cohort Study (MACS; n=32) and the Women's Interagency HIV Study (WIHS; n=32). Both sexes had high ADCC titers against HIV-1 gp120: 34.4% (n=11) and 40.6% (n=13) of men and women, respectively, had titers of 10,000; 62.5% (n=20) and 56.3% (n=18) had titers of 100,000. Groups did not differ in percent specific release (% SR), lytic units (LU), correlations of titer to viral load, or titer to CD4(+) T cells in men or women. Both groups also had similar cross-clade ADCC antibody responses (p0.5 for % SR and LU). Comparable groups of asymptomatic HIV-1-infected men and women had comparable HIV-1 gp120 ADCC antibodies. Both sexes had significant cross-clade reactivity. Differences between men and women may become evident as disease progresses; this should be evaluated at later stages of HIV-1 infection.

Although HLA-B*57 (B57) is associated with slow progression to disease following HIV-1 infection, B57 heterozygotes display a wide spectrum of outcomes, including rapid progression, viremic slow progression, and elite control. Efforts to identify differences between B57-positive (B57 + ) slow progressors and B57 + rapid progressors have largely focused on cytotoxic T lymphocyte (CTL) phenotypes and specificities during chronic stages of infection. Although CTL responses in the early months of infection are likely to be the most important for the long-term rate of HIV-1 disease progression, few data on the early CTL responses of eventual slow progressors have been available. Utilizing the Multicenter AIDS Cohort Study (MACS), we retrospectively examined the early HIV-1-specific CTL responses of 14 B57 + individuals whose time to development of disease ranged from 3.5 years to longer than 25 years after infection. In general, a greater breadth of targeting of epitopes from structural proteins, especially Gag, as well as of highly conserved epitopes from any HIV-1 protein, correlated with longer times until disease. The single elite controller in the cohort was an outlier on several correlations of CTL targeting and time until disease, consistent with reports that elite control is typically not achieved solely by protective HLA-mediated CTLs. When targeting of individual epitopes was analyzed, we found that early CTL responses to the IW9 (ISPRTLNAW) epitope of Gag, while generally subdominant, correlated with delayed progression to disease. This is the first study to identify early CTL responses to IW9 as a correlate of protection in persons with HLA-B*57.

The role of human immunodeficiency virus type 1 (HIV-1) accessory genes in pathogenesis has remained unclear because of the lack of a suitable in vivo model. The most controversial of these genes is nef. We investigated the requirement for Nef for in vivo replication and pathogenicity of two isolates of HIV-1 (HIV-1JR-CSF and HIV-1NL4-3) in human fetal thymus and liver implants in severe combined immunodeficient mice. HIV-1JR-CSF and HIV-1NL4-3 differ in their in vitro phenotypes in that HIV-1JR-CSF does not induce syncytia and is relatively noncytopathic, while HIV-1NL4-3 is highly cytopathic and readily induces syncytia. The nef mutants of both isolates grew with kinetics similar to those of parental virus strains in stimulated peripheral blood lymphocytes but demonstrated attenuated growth properties in vivo. HIV-1NL4-3 induced severe depletion of human thymocytes within 6 weeks of infection, whereas its nef mutant did not. Thus, HIV-1 Nef is required for efficient in vivo viral replication and pathogenicity.

We have examined infection of the thymus during congenitally acquired chronic lymphocytic choriomeningitis virus (LCMV) infection of mice, a classic model of antigen-specific T-cell tolerance. Our results show that (i) infection starts at the fetal stage and is maintained throughout adulthood, and (ii) this chronic infection of the thymus can be eliminated by transfer of virus-specific cytotoxic T lymphocytes (CTL) that infiltrate the thymus and clear all viral products from both medullary and cortical regions. Elimination of virus from the thymus results in abrogation of tolerance. During the fetal stage, the predominant cell type infected is the earliest precursor of T cells with a surface phenotype of Thy1+ CD4- CD8- J11d+. In the adult thymus, infection is confined primarily to the cortisone-resistant thymocytes present in the medullary region. The infected cells are CD4+ and J11d+. The presence of J11d, a marker usually associated with immature thymocytes, on infected single positive CD4+ "mature" thymocytes is intriguing and suggests that infection by this noncytolytic virus may affect development of T cells. There is minimal infection of the CD8+ medullary thymocytes or of the double positive (CD4+ CD8+) cells present in the cortex. Infection within the cortex is confined to the stromal cells. Interestingly, there is infection of the double negative (CD4- CD8-) thymocytes in the adult thymus, showing that even during adulthood the newly developing T cells are susceptible to infection by LCMV. Virus can be eliminated from the thymuses of these carrier mice by adoptive transfer of medullary region first and then from the thymic cortex. This result clearly shows the need to reevaluate the widely held notion that mature T cells are unable to reenter the thymus. In fact, in our experiments the donor T cells made up to 20 to 30% of the total cells in the thymus at 5 to 7 days after the transfer. The number of donor T cells declined as virus was eliminated from the thymus, and at 1 month posttransfer, the donor T cells were hardly detectable. The results of this study examining the dynamics of viral infection and clearance from the thymus, the primary site of T-cell development, have implications for understanding tolerance induction in chronic viral infections.

Regulatory T cells (Tregs) may play an important role in the immunopathology of chronic HIV-1 infection due to their potent suppressive activity of both T cell activation and effector function. To investigate the correlation between Tregs and immune activation during untreated chronic HIV-1 infection, we conducted a nested case-control study within the Multicenter AIDS Cohort Study (MACS). Twenty HIV-1-infected fast progressors (FP) and 40 slow progressors (SP) were included in our study using risk-set sampling. Nine age-matched HIV-1-uninfected men (UI) were also included. Cryopreserved peripheral blood mononuclear cells (PMBCs) were tested using flow cytometry analyses. We identified Tregs as Foxp3+CD25+CD4+ T cells and assessed the activation of CD4+ and CD8+ T cells by the expression of CD38, HLADR, or both markers simultaneously. There is a relative expansion of Tregs during HIV-1 infection, which is associated with disease progression. The increased CD38 expression on both CD4+ and CD8+ T cells expressed as either percentage or median fluorescence intensity (MFI) and the elevated proportion of CD8+ T cells that is HLADR+CD38+ were all associated with rapid HIV-1 progression. Counter to the assumed role of Tregs as the suppressors of activation, the expansion of Tregs was positively correlated with CD4+ T cell activation among HIV-1-infected fast progressors. The high level of Tregs associated with rapid HIV progression may suggest a detrimental role of these cells in the immune control of HIV-1 infection.

Foxp3 plays a critical role in development of CD4+ regulatory T lymphocytes (Tregs). It was originally proposed as a specific marker for Tregs, but recent studies have shown that Foxp3 can be expressed in proliferating CD8+ and CD4+ T lymphocytes. We further investigated the association between Foxp3 expression and proliferation of peripheral blood CD4+ and CD8+ T lymphocytes and focused on virus-specific memory CD8+ T lymphocytes. We found that resting peripheral blood bulk and cytomegalovirus- or HIV-1-specific CD8+ T lymphocytes do not normally express Foxp3. However, stimulation in vitro triggered these cells to express Foxp3 as well as CD25, and the addition of interleukin-2 possibly enhanced the expression of Foxp3. These data demonstrate that proliferation itself is sufficient to induce the Treg-like phenotype. Given that others have demonstrated Treg functional activity in such "induced Tregs," these results suggest that virus-specific CD8+ T lymphocytes have the capacity to acquire regulatory functions. Although the implications of Foxp3 expression in virus-specific CD8+ T lymphocytes in the immunologic control of persistent HIV-1 viremia remain to be determined, our results are consistent with Foxp3 expression playing an essential role in regulation of cell proliferation and functional outcomes for HIV-1-specific CD8+ T lymphocytes.

CD1d-restricted natural killer T (iNKT) cells are increasingly recognized as key immunoregulatory cells linking innate and adaptive immunity. These fall into functionally distinct CD4+ versus CD4- subsets that are believed to steer cellular immunity toward tolerigenic/atopic versus proinflammatory phenotypes, respectively. Preferential depletion of the CD4+ subset has been observed in HIV-1 infection, but the repletion of these cells after antiretroviral therapy has not been examined in detail. T lymphocytes, CD8+ lymphocyte activation, viremia, and iNKT cell subsets in peripheral blood were compared between 18 HIV-1-uninfected (Control) and 18 seropositive (SP) men initially not on suppressive antiretroviral therapy. Compared to the Control group, the SP group demonstrated reduction of CD4+ and lesser reduction of CD4- iNKT cells at baseline. After initiation of suppressive antiretroviral treatment, the SP CD4+ iNKT cell levels remained unchanged after a year and increased by 2 years, while CD4+ iNKT cells showed a gradual increase notable after the first year. Over the first year of treatment, there was a significant correlation between changes in total CD4+ T lymphocyte and changes in CD4+ iNKT cell levels, and a significant inverse correlation between changes in CD8+ T lymphocyte activation and changes in CD4- iNKT cell levels. These results confirm preferential depletion of tolerigenic/atopic CD4+ iNKT cells by HIV-1, and suggest that disproportionate persistence of proinflammatory CD4- iNKT cells could contribute to the inappropriate immune activation believed to cause immunodeficiency in HIV-1 infection.

Vaccines designed to induce cell-mediated immune responses against human immunodeficiency virus (HIV)-1 are being developed. Such vaccines are unlikely to provide sterilizing immunity but may be associated with reduced viral set points after infection. We modeled the potential impact of a vaccine that reduces viral set point after infection, using natural history data from 311 HIV-1 seroconverters. Log-normal parametric regression models were used to estimate the log median time to events of interest. Relative times were estimated for those with viral load set points of 30,000 copies/mL (reference group) versus those with lower viral set points. The time to key clinical events in the course of HIV-1 disease progression was significantly extended for those with viral set points 0.5-1.25 log 10 copies/mL lower than the reference group. By quantifying the anticipated clinical benefits associated with a reduction in viral set point, these findings support the use of virologic end points in HIV-1 vaccine trials.

Gut-associated lymphoid tissue is the major reservoir of lymphocytes and human immunodeficiency virus type 1 (HIV-1) replication in vivo, yet little is known about HIV-1-specific CD8+T-lymphocyte (CTL) responses in this compartment. Here we assessed the breadth and magnitude of HIV-1-specific CTL in the peripheral blood and sigmoid colon mucosa of infected subjects not on antiretroviral therapy by enzyme-linked immunospot analysis with 53 peptide pools spanning all viral proteins. Comparisons of blood and mucosal CTL revealed that the magnitude of pool-specific responses is correlated within each individual (meanr2= 0.82 ± 0.04) and across all individuals (r2= 0.75;P< 0.001). Overall, 85.1% of screened peptide pools yielded concordant negative or positive results between compartments. CTL targeting was also closely related between blood and mucosa, with Nef being the most highly targeted (mean of 2.4 spot-forming cells [SFC[/106CD8+T lymphocytes/amino acid [SFC/CD8/aa]), followed by Gag (1.5 SFC/CD8/aa). Finally, comparisons of peptide pool responses seen in both blood and mucosa (concordant positives) versus those seen only in one but not the other (discordant positives) showed that most discordant results were likely an artifact of responses being near the limit of detection. Overall, these results indicate that HIV-1-specific CTL responses in the blood mirror those seen in the mucosal compartment in natural chronic infection. For protective or immunotherapeutic vaccination, it will be important to determine whether immunity is elicited in the mucosa, which is a key site of initial infection and subsequent HIV-1 replication in vivo.

Sequential infection with different strains of human immunodeficiency virus type 1 (HIV-1) is a rarely identified phenomenon with important implications for immunopathogenesis and vaccine development. Here, we identify an individual whose good initial control of viremia was lost in association with reduced containment of a superinfecting strain. Subject 2030 presented with acute symptoms of HIV-1 infection with high viremia and an incomplete seroconversion as shown by Western blotting. A low set point of viremia (∼1,000 HIV-1 copies/ml) was initially established without drug therapy, but a new higher set point (∼40,000 HIV-1 copies/ml) manifested about 5 months after infection. Drug susceptibility testing demonstrated a multidrug-resistant virus initially but a fully sensitive virus after 5 months, and an analysis of pol genotypes showed that these were two phylogenetically distinct strains of virus (strains A and B). Replication capacity assays suggested that the outgrowth of strain B was not due to higher fitness conferred by pol , and env sequences indicated that the two strains had the same R5 coreceptor phenotype. Delineation of CD8 + -T-lymphocyte responses against HIV-1 showed a striking pattern of decay of the initial cellular immune responses after superinfection, followed by some adaptation of targeting to new epitopes. An examination of targeted sequences suggested that differences in the recognized epitopes contributed to the poor immune containment of strain B. In conclusion, the rapid overgrowth of a superinfecting strain of HIV-1 of the same subtype raises major concerns for effective vaccine development.

To investigate possible mechanisms behind HIV-1 escape from CTL, we performed detailed longitudinal analysis of Gag (SLYNTVATL)- and RT (ILKEPVHGV)-specific CTL responses and plasma epitope sequences in five individuals. Among those with CTL against consensus epitope sequences, epitope mutations developed over several years, invariably followed by decay of the CTL targeting the consensus epitopes. The maturation state of the CTL varied among individuals and appeared to affect the rate of epitope mutation and CTL decay, despite similar IFN-γ production. Escape mutations were oligoclonal, suggesting fitness constraints. The timing of escape indicated that the net selective advantage of escape mutants was slight, further underscoring the importance of understanding factors determining selective pressure and viral fitness in vivo. Our data show surprisingly consistent decay of CTL responses after epitope escape mutation and provide insight into potential mechanisms for both immune failure and shifting CTL specificities.

The use of reporter constructs of HIV-1 molecular clones has been useful in studying the effects of infection on individual cells. A strategy of producing viral half-genome constructs containing reporter genes that can be used to produce recombinant complete infectious virions is described. This approach allows rapid generation of reporter and non-reporter virions from the same genetic construct, obviating the need to produce a new reporter genetic construct for each viral change to be studied. We provide an example of the utility of this system for studying the effects of Nef on infected cells.

An important requisite for full immune reconstitution following drug or hematopoietic stem cell gene therapy treatments of human immunodeficiency virus type 1 (HIV-1) infection is that precursor cells must be able to differentiate via the T-lymphoid pathway and give rise to naive T cells capable of responding to antigens. Although most adult patients treated with highly active antiretroviral therapy (HAART) experience an increase in CD4+ T-cell counts associated with a decrease in viral load in the peripheral blood (4, 13, 27–29, 36), much of this rise is secondary to an expansion of cells in the peripheral lymphoid compartment rather than de novo thymic output (4, 27). HIV-1 infection gives rise to pathology in the thymus, which is manifested in part by a severe depletion of double-positive (DP) CD4/CD8 cortical thymocytes (31). The degree of thymic dysfunction in pediatric HIV-1-infected patients correlates with progression to AIDS and survival (19). The thymus is known to involute in healthy adults (32, 34). However, ongoing thymopoiesis can be demonstrated in the adult thymus (5) and de novo T-cell development can occur in adults following T-cell depletion states, such as myelosuppression (11, 22) and HIV-1 infection following 4 to 6 months of HAART (4, 27). Radiographically measured increases in thymic tissue size have been correlated with high total and naive circulating CD4+ lymphocyte counts in HIV-1-infected adults (23). New findings indicate that the levels of detectable thymic emigrants increase rapidly in the periphery following HAART (9). Together, these data seem to indicate that a degree of immune reconstitution that is associated with de novo T-cell development occurs even in adult HIV-1-infected patients after viral replication is suppressed and that the thymus may play an important role in this process. However, it is unclear whether this partial immune restoration and de novo T-cell development observed after prolonged HAART can be sustained for a long period. The severe combined immunodeficient (SCID) mouse implanted with human fetal liver and thymus (SCID-hu) has been employed as an in vivo system to study T-cell development and HIV-1 pathogenesis (1, 6, 14, 24–26). Using this model, we demonstrated that following antiretroviral therapy of animals bearing thymic implants severely depleted of human thymocytes by HIV-1 infection, renewed thymopoiesis occurs from both endogenous and exogenous precursors, indicating that both thymic support and stem cell functions are preserved after exposure to a high viral burden (37). However, despite continuing antiretroviral therapy, eventual thymocyte depletion ensues in this system. Our preliminary assessment of this eventual decline included measurements of proviral burden in thymocytes and viremia of treated and untreated animals (37). These experiments suggested that complete control of viral replication was not achieved and that viral breakthrough may have contributed, at least partially, to the eventual thymic failure following therapy. If viral replication is proven to remain in check during the period of eventual T-cell decline, other reasons such as cessation of thymic stromal support function or direct or indirect compromise of thymic progenitors would need to be invoked to explain the short-lived nature of T-cell renewal. Such scenarios would have important implications for the application of pharmacology-, stem cell-, and gene-based treatment approaches to HIV-1 infection. To study the kinetics of virus expression in thymocytes in the context of antiretroviral therapy, we used an HIV-1 reporter construct that is pathogenic in vivo and induces expression of the murine CD24 surface marker on infected cells. Thus, the kinetics of virus expression and thymocyte resurgence and decline can be studied in this system by flow cytometry (15). Despite combination therapy, we found an increase in the percentage of cells expressing virus during the late stages of infection, coincident with a second decline in thymocytes. These data indicate that viral breakthrough is associated with late thymocyte decline and that renewed virus replication may be responsible for the eventual thymic failure observed in this system.

The mechanism of CD4+ cell loss in lymphoid organs is unknown. In this study, human immunodeficiency virus (HIV) infection of human fetal thymus/liver implants in severe combined immunodeficient mice was used to investigate the mechanism of HIV-induced depletion of CD4-bearing cells in vivo. The implants were assessed for depletion of CD4+ thymocytes, apoptosis, and viral burden. We detected two phases of CD4 cell depletion, an initial rapid phase and a more gradual later phase. Compared to mock-infected implants, HIV-infected implants did not demonstrate detectable increases in the levels of apoptosis while severe depletion of CD4-bearing cells was ongoing. During peak loss of CD4+ cells, high viral burden was observed, suggesting that loss of CD4+ cells in this in vivo system is due to direct killing of infected thymocytes. Increased levels of apoptosis were observed during the later phase of thymocyte depletion; however, these apoptotic cells lacked CD4. This finding suggests that a second indirect mechanism may be responsible for the destruction of CD4- CD8+ thymocytes in vivo. Taken together, these results suggest that CD4+ and CD4- cells may die by different mechanism(s).

Objective : Understanding the interaction between HIV and developing thymocytes is crucial in determining how HIV infection perturbs the immune system. We determined which thymocyte subsets can harbor and express HIV. Design : HIV expression in mature and immature thymocytes obtained from surgical specimens from non-infected children was determined after in vitro infection with the syncytium-inducing, cytopathic NL4-3 and the non-syncytium-inducing, relatively noncytopathic JR-CSF isolates. Methods : Intracellular staining for the HIV p24 gag antigen was combined with cell surface phenotyping to determine thymocyte subsets expressing HIV. Infection was quantitated by polymerase chain reaction on sorted subsets. Results : NL4-3 replicated faster and to higher titers and caused a more severe decrease of all CD4-bearing thymocytes than did JR-CSF. In addition, both immature CD1+ and mature CD1- thymocytes expressed NL4-3, whereas only mature CD1- cells expressed JR-CSF. The tropism of NL4-3 for these immature cells suggests a mechanism for a more profound impact on T-cell maturation than that seen with JR-CSF. We also found that thymocytes lacking cell surface CD4 (CD4-CD8- and CD4-CD8+ subsets) expressed virus with either isolate late in infection, when viral levels were high. The CD4-CD8- cells expressing HIV were mature CD3 bright T-cell receptor (TCR)α/β bright cells. Conclusions : These results show that NL4-3 can be expressed by thymocytes at immature and mature stages of differentiation and cause severe loss of CD4+ cells. Thus, tropism of a virus for immature cells can affect the capability of the thymus to produce new T lymphocytes leading to a greater impact on development and functions of the immune system. It is proposed that this in vitro model can be used to study pathogenic mechanisms in the thymus.

We have investigated the in vivo pathogenic properties of two molecularly cloned strains of human immunodeficiency virus type 1 (HIV-1), HIV-1NL4-3 and HIV-1JR-CSF, in human fetal thymus/liver implants in severe combined immunodeficient mice. Studies comparing their in vivo replication kinetics and abilities to induce CD4+ thymocyte depletion were performed. HIV-1NL4-3 replicated in vivo with faster kinetics and induced greater levels of CD4+ thymocyte depletion than did HIV-1JR-CSF. These results demonstrate that different viral isolates have different pathogenic properties in this system. In the SCID-hu model, this pathogenesis most likely occurs in the absence of an immune response. Therefore, we investigated whether the absence of immune selection resulted in extensive genetic variation and the generation of viral quasispecies. To this end, DNA corresponding to the fourth variable domain region of the viral envelope gp120 protein recovered from biopsy samples at 6 weeks postinfection was sequenced. Little genetic variation was noted in either HIV-1JR-CSF- or HIV-1NL4-3-infected implants. The mutation levels demonstrated in both viral strains were more reflective of the acute rather than the chronic phase of HIV-1 infection in humans. These results suggest that the SCID-hu mouse model can be used to study the in vivo pathogenicity of different HIV-1 isolates in the absence of host immune selective pressures.

This study documents that virus-specific CTL can persist indefinitely in vivo. This was accomplished by transferring Thy-1.1 T cells into Thy-1.2 recipient mice to specifically identify the donor T cell population and to characterize its antigenic specificity and function by using a virus-specific CTL assay. Thy-1.1+ T cells from mice previously immunized with lymphocytic choriomeningitis virus (LCMV) were transferred into Thy-1.2 mice persistently infected with LCMV. The transferred LCMV-specific CTL (Thy-1.1+ CD8+) eliminate virus from the chronically infected carriers and persist in the recipient mice in small numbers, comprising only a minor fraction of the total T cells. Upon re-exposure to virus, these long-lived "resting" CD8+ T cells proliferate in vivo to become the predominant cell population. These donor CD8+ T cells can be recovered up to a year post-transfer and still retain antigenic specificity and biological function. They kill LCMV infected H-2-matched cells in vitro and can eliminate virus upon transfer into a second infected host. In addition, these long-lived CD8+ T cells appear not to be dependent on help from CD4+ T cells, since depletion of CD4+ T cells has minimal or no effect on their biological properties (proliferation, CTL response, viral clearance). These donor CTL also exhibit an immunodominance over the host-derived LCMV-specific CTL response. When both host and donor T cells are present, the donor CTL response is dominant over the potential CTL response of the cured carrier host. Taken together, these results suggest that virus-specific CTL can persist for the life span of the host as memory cells.

Data in this manuscript were collected by the MACS, with centers (Principal Investigators) at the Johns Hopkins University Bloomberg School of Public Health (Joseph B. Margolick, Lisa Jacobson); Howard Brown Health Center and Northwestern University Medical School (John Phair); University of California, Los Angeles (Roger Detels, Beth Jamieson); and University of Pittsburgh (Charles Rinaldo). The MACS is funded by the National Institute of Allergy and Infectious Diseases, with additional supplemental funding from the National Cancer Institute. UO1-AI-35042, 5-MO1-RR-00722 (GCRC), UO1-AI-35043, UO1-AI-37984, UO1-AI-35039, UO1-AI-35040, UO1-AI-37613, UO1-AI-35041. The website is located at http://www.statepi.jhsph.edu/macs/macs.html. I.J.W. is funded by the Covance Fellowship of the Royal Australasian College of Physicians.

The lifelong chronic lymphocytic choriomeningitis virus (LCMV) infection established in neonatally or congenitally infected mice can be eliminated by adoptive transfer of lymphoid cells from LCMV-immune mice. In this study, we have identified the effector cells mediating the clearance of persistent and disseminated LCMV infection. Using mice that are recombinant in the H-2 region and by selective depletion of lymphocyte subpopulations, we show that viral clearance was mediated by LCMV-specific Lyt2+ L3T4- T cells that are restricted to the class I genes of the major histocompatibility complex. In addition, our results show a requirement for host-derived bone marrow cells for the effective elimination of virus from the liver. These studies emphasize the importance of virus-specific T cells and an intact bone marrow function in viral clearance.

The mechanism of viral clearance was studied by using the mouse model of chronic infection with lymphocytic choriomeningitis virus. Distinct patterns of viral clearance and histopathology were observed in different organs after adoptive immune therapy of persistently infected (carrier) mice. Clearance from the liver occurred within 30 days and was accompanied by extensive mononuclear cell infiltrates and necrosis of hepatocytes. Infectious virus and viral antigen were eliminated concurrently. This pattern of viral clearance was also seen in most other tissues (i.e., lung, spleen, lymph nodes, pancreas, etc.). In contrast, a different pattern of clearance was observed in the brain. Infectious virus was eliminated within 30 days, but viral antigen persisted in the central nervous systems of treated carrier mice for up to 90 days. The urinary system was the most resistant to immune therapy. Elimination of infectious virus and viral antigen from the kidney took greater than 200 days and even then was not complete; trace levels of infectious virus were still present in the kidneys of some treated carrier mice. After immune therapy, viral antigen in the kidney was located within renal tubules that costained for intracellular mouse immunoglobulin G. This unusual staining pattern, coupled with the observation of large numbers of plasma cells within the kidney, suggests that virus-immunoglobulin G complexes found in the tubules may represent in situ immune complex formation as opposed to deposition of circulating immune complexes. In conclusion, these results suggest that the site (organ) of viral persistence is an important consideration in developing treatment strategies for controlling chronic viral infections.