Abrogation of in vitro suppression of human immunodeficiency virus type 1 (HIV-1) replication mediated by CD8+ T lymphocytes of asymptomatic HIV-1 carriers by staphylococcal enterotoxin B and phorbol esters through induction of tumor necrosis factor alpha.

CD8+ T lymphocytes of asymptomatic human immunodeficiency virus type 1 (HIV-1) carriers (AC) suppress HIV-1 replication in vitro. Failure of host defense mechanisms and increased virus proliferation are associated with disease progression. The exact mechanisms inducing these changes at the advanced stage of the disease are still obscure. In this study, we searched for experimental conditions favoring the abrogation of the suppression of viral replication in peripheral blood mononuclear cells (PBMC) of AC by using various pharmacological and biological probes modifying cell activation. Among such agents, staphylococcal enterotoxin B (SEB) and phorbol 12-myristate 13-acetate (PMA) markedly increased otherwise low levels of HIV-1 replication in cultures of phytohemagglutinin-stimulated AC PBMC following in vitro HIV-1 LAI infection. A similar but less pronounced virus induction was also observed in macrophage-tropic HIV-1. Individual pretreatment of CD4+ and CD8+ PBMC fractions with these agents caused a reduction in CD8+ cell proliferation and enhanced HIV-1 replication in CD4+ cells. SEB- and PMA-mediated augmentation of HIV-1 replication in AC PBMC was significantly blocked by neutralizing antibody to tumor necrosis factor-alpha (TNF-alpha), although recombinant TNF-alpha alone failed to reproduce the effects of SEB or PMA. Our results suggest that the induction of TNF-alpha may be one of the mechanisms that overcomes the CD8+-induced suppression of HIV-1 replication in AC and that it may induce HIV-1 replication.     

Gamma interferon-induced, nitric oxide-mediated inhibition of vaccinia virus replication.

Gamma interferon (IFN-gamma)-induced nitric oxide synthase (iNOS) and nitric oxide (NO) production in the murine macrophage-like RAW 264.7 cells were previously shown to inhibit the replication of the poxviruses vaccinia virus (VV) and ectromelia virus and herpes simplex virus type 1. In the current study, we performed biochemical analyses to determine the stage in the viral life cycle blocked by IFN-gamma-induced NO. Antibodies specific for temporally expressed viral proteins, a VV-specific DNA probe, and transmission electron microscopy were used to show that the cytokine-induced NO inhibited late protein synthesis, DNA replication, and virus particle formation but not expression of the early proteins analyzed. Essentially similar results were obtained with hydroxyurea and cytosine arabinoside, inhibitors of DNA replication. Enzymatically active iNOS was detected in the lysates of IFN-gamma-treated but not in untreated RAW 264.7 cells. The IFN-gamma-treated RAW 264.7 cells which express iNOS not only were resistant to productive infection but also efficiently blocked the replication of VV in infected bystander cells of epithelial origin. This inhibition was arginine dependent, correlated with nitric production in cultures, and was reversible by the NOS inhibitor N omega-monomethyl-L-arginine.     

Nitric oxide regulates MIP-1alpha expression in primary macrophages and T lymphocytes: implications for anti-HIV-1 response

BACKGROUND: Chemokines and chemokine receptors have been shown to play a critical role in HIV infection. Chemokine receptors have been identified as coreceptors for viral entry into susceptible target cells, and several members of the beta chemokine subfamily of cytokines, MIP-1alpha, MIP-1beta, and RANTES, have been identified as the major human immunodeficiency virus (HIV)-suppressive factors produced by activated CD8+ T lymphocytes. In macrophages, HIV-1 infection itself was shown to upregulate the production of MIP-1alpha and MIP-1beta. In the present study, we address the mechanisms by which HIV-1 infection regulates beta chemokine responses in macrophages and lymphocytes. MATERIAL AND METHODS: To address whether nitric oxide (NO), generated as a consequence of HIV-1 infection, regulates beta chemokine responses in monocyte/macrophages and/or macrophage-depleted peripheral blood mononuclear cells (PBMCs) these two cell populations were isolated from HIV seronegative donors, placed in culture, and infected with HIV-1 in either the presence or absence of exogenous activators (e.g. lipopolysaccharide, phytohemagglutinin), inhibitors of nitric oxide synthase (NOS), or chemical donors of NO. Cultures were analyzed for beta chemokine responses by ELISA and RNase protection. RESULTS: LPS-induced MIP-1alpha release is enhanced in HIV-1-infected, as compared to uninfected, monocyte/macrophage cultures, and this enhancing effect is partially blocked by the addition of inhibitors of NOS, and can be reproduced by chemical generators of NO even in the absence of HIV-1 infection. A similar strategy was used to demonstrate a role for NO in HIV-1-mediated induction of MIP-1alpha in unstimulated macrophage cultures. NOS inhibitors also decreased MIP-1alpha and MIP-1beta production by phytohemagglutinin-stimulated monocyte-depleted PBMC cultures. CONCLUSIONS: These results indicate that NO amplifies MIP-1alpha responses in activated macrophages and lymphocytes, and suggests that this pleiotropic molecule might function as an enhancing signal that regulates secretion of beta chemokines during HIV-1 infection. These findings reveal a novel mechanism by which NO might regulate the anti-HIV activity of immune cells.     

Reversible S-nitrosation and inhibition of HIV-1 protease.

Nitric oxide (*NO) is a short-lived free radical with many functions including vasoregulation, synaptic plasticity, and immune modulation and has recently been associated with AIDS pathology. Various pathophysiological conditions, such as viral infection, trigger inducible nitric oxide synthase (iNOS) to synthesize NO in the cell. NO-derived species can react with thiols of proteins and form nitrosothiol adducts. HIV-1 protease (HIV-PR) contains two cysteine residues, Cys67 and Cys95, which are believed to serve a regulatory function. We have found that HIV-PR is inactivated by nitric oxide produced in vitro by NO donors and by iNOS. Sodium nitroprusside inhibited HIV-PR by 70%, and S-nitroso-N-acetylpenicillamine completely inhibited the enzyme. Furthermore, iNOS generated sufficient NO to inhibit HIV-PR activity by almost 90%. This inactivation was reversed by the addition of reducing agents. Treatment of HIV-PR with NO donors and ritonavir (a competitive peptide inhibitor) indicates that NO exerts its effect through a site independent of the active site of HIV-PR. Using electrospray ionization mass spectrometry, we found that NO forms S-nitrosothiols on Cys67 and Cys95 of HIV-PR which directly correlate with a loss of activity. These data indicate that NO may suppress HIV-1 replication by directly inhibiting HIV-PR.     

Nitric oxide synthesis enhances human immunodeficiency virus replication in primary human macrophages

Macrophages are suspected to play a major role in human immunodeficiency virus (HIV) infection pathogenesis, not only by their contribution to virus dissemination and persistence in the host but also through the dysregulation of immune functions. The production of NO, a highly reactive free radical, is thought to act as an important component of the host immune response in several viral infections. The aim of this study was to evaluate the effects of HIV type 1 (HIV-1) Ba-L replication on inducible nitric oxide synthase (iNOS) mRNA expression in primary cultures of human monocyte-derived macrophages (MDM) and then examine the effects of NO production on the level of HIV-1 replication. Significant induction of the iNOS gene was observed in cultured MDM concomitantly with the peak of virus replication. However, this induction was not accompanied by a measurable production of NO, suggesting a weak synthesis of NO. Surprisingly, exposure to low concentrations of a NO-generating compound (sodium nitroprusside) and L-arginine, the natural substrate of iNOS, results in a significant increase in HIV replication. Accordingly, reduction of L-arginine bioavailability after addition of arginase to the medium significantly reduced HIV replication. The specific involvement of NO was further demonstrated by a dose-dependent inhibition of viral replication that was observed in infected macrophages exposed to N(G)-monomethyl L-arginine and N(G)-nitro-L-arginine methyl ester (L-NAME), two inhibitors of the iNOS. Moreover, an excess of L-arginine reversed the addition of L-NAME, confirming that an arginine-dependent mechanism is involved. Finally, inhibitory effects of hemoglobin which can trap free NO in culture supernatants and in biological fluids in vivo confirmed that endogenously produced NO could interfere with HIV replication in human macrophages.     

A critical role of nitric oxide in human immunodeficiency virus type 1-induced hyperresponsiveness of cultured monocytes.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) infection leads to a general exhaustion of the immune system. Prior to this widespread decline of immune functions, however, there is an evident hyperactivation of the monocyte/macrophage arm. Increased levels of cytokines and other biologically active molecules produced by activated monocytes may contribute to the pathogenesis of HIV disease both by activating expression of HIV-1 provirus and by direct effects on cytokine-sensitive tissues, such as lung or brain. In this article, we investigate mechanisms of hyperresponsiveness of HIV-infected monocytes. MATERIALS AND METHODS: The study was performed on monocyte cultures infected in vitro with a monocytetropic strain HIV-1ADA. Cytokine production was induced by stimulation of cultures with lipopolysaccharides (LPS) and measured by ELISA. To study involvement of nitric oxide (NO) in the regulation of cytokine expression, inhibitors of nitric oxide synthase (NOS) or chemical donors of NO were used. RESULTS: We demonstrate that infection with HIV-1 in vitro primes human monocytes for subsequent activation with LPS, resulting in increased production of pro-inflammatory cytokines tumor necrosis factor (TNF) and interleukin 6 (IL-6). This priming effect can be blocked by Ca(2+)-chelating agents and by the NOS inhibitor L-NMMA, but not by hemoglobin. It could be reproduced on uninfected monocyte cultures by using donors of NO, but not cGMP, together with LPS. CONCLUSIONS: NO, which is expressed in HIV-1-infected monocyte cultures, induces hyperresponsiveness of monocytes by synergizing with calcium signals activated in response to LPS stimulation. This activation is cGMP independent. Our findings demonstrate the critical role of NO in HIV-1-specific hyperactivation of monocytes.     

Macrophage control of herpes simplex virus type 1 replication in the peripheral nervous system

After corneal infection, herpes simplex virus type 1 (HSV-1) invades sensory neurons with cell bodies in the trigeminal ganglion (TG), replicates briefly, and then establishes a latent infection in these neurons. HSV-1 replication in the TG can be detected as early as 2 days after corneal infection, reaches peak titers by 3-5 days after infection, and is undetectable by 7-10 days. During the period of HSV-1 replication, macrophages and gammadelta TCR+ T lymphocytes infiltrate the TG, and TNF-alpha, IFN-gamma, the inducible nitric oxide synthase (iNOS) enzyme, and IL-12 are expressed. TNF-alpha, IFN-gamma, and the iNOS product nitric oxide (NO) all inhibit HSV-1 replication in vitro. Macrophage and gammadelta TCR+ T cell depletion studies demonstrated that macrophages are the main source of TNF-alpha and iNOS, whereas gammadelta TCR+ T cells produce IFN-gamma. Macrophage depletion, aminoguanidine inhibition of iNOS, and neutralization of TNF-alpha or IFN-gamma all individually and synergistically increased HSV-1 titers in the TG after HSV-1 corneal infection. Moreover, individually depleting macrophages or neutralizing TNF-alpha or IFN-gamma markedly reduced the accumulation of both macrophages and gammadelta TCR+ T cells in the TG. Our findings establish that after primary HSV-1 infection, the bulk of virus replication in the sensory ganglia is controlled by macrophages and gammadelta TCR+ T lymphocytes through their production of antiviral molecules TNF-alpha, NO, and IFN-gamma. Our findings also strongly suggest that cross-regulation between these two cell types is necessary for their accumulation and function in the infected TG.     

Quantitation of a lentivirus in its natural host: simian immunodeficiency virus in African green monkeys.

We have examined the viral load in the peripheral blood of simian immunodeficiency virus (SIV)-infected African green monkeys with a view to the unexplained apathogenicity of African green monkey SIV (SIVagm) in its natural host. By using polymerase chain reaction, viral DNA was detected in fresh peripheral blood mononuclear cells (PBMC) of each of nine seropositive animals. The virus DNA load was variable among the monkeys tested, ranging from 5 to 50 (mean = 15) copies per 10(5) PBMC, which is comparable to that of human immunodeficiency virus type 1 (HIV-1) in humans. The level of infectious SIVagm in PBMC was measured by endpoint dilution cultures. SIVagm was recovered from PBMC from 14 of 17 antibody-positive monkeys (82%), and the mean SIVagm titer in PBMC of seropositive African green monkeys was 10 tissue culture infectious doses per 10(6) cells, similar to the titer shown for HIV in asymptomatic carriers. Free infectious virus was isolated from the plasma of 4 of 17 monkeys (24%), and SIVagm expression in peripheral blood in vivo, as demonstrated by in situ hybridization, was detectable only in those animals which were viremic. SIVagm replication is therefore not totally suppressed in vivo, and SIVagm has a viral load equivalent to that seen for HIV-1 in asymptomatic humans.     

Undetectable levels of tumor necrosis factor-alpha,nitric oxide and inadequate expression of inducible nitric oxide synthase in congenital hypothyroidism

OBJECTIVE: To analyse the production of TNF-alpha and NO in euthyroid and hypothyroid newborns. PATIENTS: A cross-sectional study was conducted involving 10 newborns diagnosed with primary congenital hypothyroidism (CH; group A) and 10 euthyroid children (group B). RESULTS: There were undetectable plasma levels of TNF-alpha and NO in the hypothyroid children, however plasma levels of TNF-alpha (5.5 0.5 pg/ml) and NO (5.6 1.7 microM) were detected at normal levels in all euthyroid children. Moreover, expression of iNOS mRNA in PBMC, determined by RT-PCR, was negative in both groups of infants. CONCLUSION: TNF-alpha and NO production are both impaired in hypothyroid newborns. We report for the first time evidence of undetectable levels of TNF-alpha and NO in infants with CH.     

Identification of human endogenous retrovirus-specific T cell responses in vertically HIV-1-infected subjects

Human endogenous retrovirus (HERV)-specific T cell responses in HIV-1-infected adults have been reported. Whether HERV-specific immunity exists in vertically HIV-1-infected children is unknown. We performed a cross-sectional analysis of HERV-specific T cell responses in 42 vertically HIV-1-infected children. HERV (-H, -K, and -L family)-specific T cell responses were identified in 26 of 42 subjects, with the greatest magnitude observed for the responses to HERV-L. These HERV-specific T cell responses were inversely correlated with the HIV-1 plasma viral load and positively correlated with CD4(+) T cell counts. These data indicate that HERV-specific T cells may participate in controlling HIV-1 replication and that certain highly conserved HERV-derived proteins may serve as promising therapeutic vaccine targets in HIV-1-infected children.     

Differences among HIV-1 variants in their ability to elicit secretion of TNF-alpha

HIV-1 infection of human PBMC has been shown to elicit secretion of several different cytokines. TNF-alpha secretion induced by this virus has been of particular interest because it has been associated with the development of HIV-1 dementia and because TNF-alpha increases viral replication by enhancing NF-kappaB interaction with the viral promoter, the HIV-1 long terminal repeat. Thus, an autocrine pathway is potentially created in which HIV-1 stimulates its own replication. Conflicting reports exist, however, on the ability of HIV-1 to induce TNF-alpha secretion in vitro or in vivo. Using experimental protocols that controlled for potential bacterial endotoxin-induced TNF-alpha secretion, the current study demonstrates significant differences in TNF-alpha-eliciting properties among primary and laboratory obtained HIV-1. The relative TNF-alpha-inducing ability of different variants is conserved when tested using PBMC from different individuals. Elicitation of TNF-alpha secretion was not blocked by exposure of cells to zidovudine, indicating that viral integration was not required to induce secretion. Rather, the interaction between the virus and cell surface is critical for TNF-alpha induction, as Abs against CD4 or CCR5 blocked the induction of TNF-alpha synthesis by PBMC when added before virus exposure. Furthermore, the ability to induce TNF-alpha secretion mapped to a region of the HIV-1 env gene that includes the third hypervariable domain. Differences in the ability of different HIV-1 variants to elicit TNF-alpha may account for individual differences in HIV-1 disease course.     

Discordance between frequency of human immunodeficiency virus type 1 (HIV-1)-specific gamma interferon-producing CD4(+) T cells and HIV-1-specific lymphoproliferation in HIV-1-infected subjects with active viral replication

One hallmark of uncontrolled, chronic human immunodeficiency virus type 1 (HIV-1) infection is the absence of strong HIV-1-specific, CD4(+) T-cell-proliferative responses, yet the mechanism underlying this T helper (Th)-cell defect remains controversial. To better understand the impact of HIV-1 replication on Th-cell function, we compared the frequency of CD4(+) Th-cell responses based on production of gamma interferon to lymphoproliferative responses directed against HIV-1 proteins in HIV-1-infected subjects with active in vivo viral replication versus those on suppressed highly active antiretroviral therapy (HAART). No statistically significant differences in the frequencies of cytokine-secreting, HIV-1-specific CD4(+) T cells between the donor groups were found, despite differences in viral load and treatment status. However, HIV-1-specific lymphoproliferative responses were significantly greater in the subjects with HAART suppression than in subjects with active viral replication. Similar levels of HIV-1 RNA were measured in T-cell cultures stimulated with HIV-1 antigens regardless of donor in vivo viral loads, but only HIV-1-specific CD4(+) T cells from subjects with HAART suppression proliferated in vitro, suggesting that HIV-1 replication in vitro does not preclude HIV-1-specific lymphoproliferation. This study demonstrates a discordance between the frequency and proliferative capacity of HIV-1-specific CD4(+) T cells in subjects with ongoing in vivo viral replication and suggests that in vivo HIV-1 replication contributes to the observed defect in HIV-1-specific CD4(+) T-cell proliferation.     

Disassociation between the in vitro and in vivo effects of nitric oxide on a neurotropic murine coronavirus.

Intranasal inoculation of the neuroattenuated OBLV60 strain of mouse hepatitis virus results in infection of mitral neurons in the olfactory bulb, followed by spread along olfactory and limbic pathways to the brain. Immunocompetent BALB/c mice were able to clear virus by 11 days postinfection (p.i.). Gamma interferon (IFN-gamma) may play a role in clearance of OBLV60 from infected immunocompetent BALB/c mice through a nonlytic mechanism. Among the variety of immunomodulatory activities of IFN-gamma is the induction of expression of inducible nitric oxide synthase (iNOS), an enzyme responsible for the production of nitric oxide (NO). Studies were undertaken to investigate the role of IFN-gamma and NO in host defense and clearance of OBLV60 from the central nervous system (CNS). Exposure of OBLV60-infected OBL21a cells, a mouse neuronal cell line, to the NO-generating compound S-nitroso-L-acetyl penicillamine resulted in a significant decrease in viral replication, indicating that NO interfered with viral replication. Furthermore, infection of IFN-gamma knockout (GKO) mice and athymic nude mice with OBLV60 resulted in low-level expression of iNOS mRNA and protein in the brains compared to that of OBLV60-infected BALB/c mice. Nude mice were unable to clear virus and eventually died between days 11 and 14 p.i. (B. D. Pearce, M. V. Hobbs, T. S. McGraw, and M. J. Buchmeier, J. Virol. 68:5483-5495, 1994); however, GKO mice survived infection and cleared virus by day 18 p.i. These data suggest that IFN-gamma production in the olfactory bulb contributed to but may not be essential for clearance of OBLV60 from the brain. In addition, treatment of OBLV60-infected BALB/c mice with aminoguanidine, a selective inhibitor of iNOS activity, did not result in any increase in mortality, and the mice cleared the virus by 11 days p.i. These data suggest that although NO was able to block replication of virus in vitro, expression of iNOS with NO release in vivo did not appear to be the determinant factor in clearance of OBLV60 from CNS neurons.     

Inhibitors of inducible nitric oxide (NO) synthase are more effective than an NO donor in reducing carbon-tetrachloride induced acute liver injury

The exact functional role of nitric oxide (NO) in liver injury is currently a source of controversy. NO is enzymatically synthesized by nitric oxide synthase (NOS). In this study, we assessed the role of inducible NOS (iNOS) in carbon tetrachloride (CCl4)-induced acute liver injury using inhibitors of iNOS, and an NO donor. Adult ICR mice were injected with CCl4 with or without the iNOS inhibitors (5-methylisothiourea hemisulfate [SMT] and l-N6-(1-iminoethyl)-lysine [L-NIL]) and an NO donor (Sodium Nitroprusside [SNP]). Blood and liver tissues were collected for analysis. Immunohistochemistry (IHC), serum alanine aminotransferase (ALT), serum total 8-isoprostane analysis, RT-PCR, Western Blotting (WB) and EMSA were done. Our results showed increased levels of ALT, necrosis, total 8-isoprostane and nitrotyrosine after CCl4 administration. iNOS inhibitors and SNP abrogated these effects but the effect was more pronounced with SMT and L-NIL. RT-PCR, WB and IHC in CCl4-treated mice demonstrated upregulation of TNF-alpha, iNOS, and COX-2. The administration of iNOS inhibitors with CCl4 diminished the expression of these proinflammatory mediators. NF-kappaB was also upregulated in CCl4-treated mice and was reversed in mice pretreated with iNOS inhibitors. SNP pretreated mice also showed a lower expression of COX-2 when compared with CCl4 treated mice but TNF-alpha, iNOS and NF-kappaB activity were unaffected. We propose that a high level of nitric oxide is associated with CCl4-induced acute liver injury and the liver injury can be ameliorated by decreasing the NO level with iNOS inhibitors and an NO donor with the former more effective in reducing CCl4-induced liver injury.     

N-acetylcysteine inhibits in vivo nitric oxide production by inducible nitric oxide synthase.

This in vivo study evaluates the effect of N-acetylcysteine (NAC) administration on nitric oxide (NO) production by the inducible form of nitric oxide synthase (iNOS). NO production was induced in the rat by the ip administration of 2 mg/100 g lipopolysaccharide (LPS). This treatment caused: (1) a decrease in body temperature within 90 min, followed by a slow return to normal levels; (2) an increase in plasma levels of urea, nitrite/nitrate, and citrulline; (3) the appearance in blood of nitrosyl-hemoglobin (NO-Hb) and in liver of dinitrosyl-iron-dithiolate complexes (DNIC); and (4) increased expression of iNOS mRNA in peripheral blood mononuclear cells (PBMC). Rat treatment with 15 mg/100 g NAC ip, 30 min before LPS, resulted in a significant decrease in blood NO-Hb levels, plasma nitrite/nitrate and citrulline concentrations, and liver DNIC complexes. PBMC also showed a decreased expression of iNOS mRNA. NAC pretreatment did not modify the increased levels of plasma urea or the hypothermic effect induced by the endotoxin. The administration of NAC following LPS intoxication (15 min prior to sacrifice) did not affect NO-Hb levels. These results demonstrate that NAC administration can modulate the massive NO production induced by LPS. This can be attributed mostly to the inhibitory effect of NAC on one of the events leading to iNOS protein expression. This hypothesis is also supported by the lack of effect of late NAC administration.     

Protective effect of radicicol against LPS/IFN-gamma-induced neuronal cell death in rat cortical neuron-glia cultures

We investigated the protective activity of radicicol, an antifungal antibiotic, against inflammation-induced neurotoxicity in neuron-glia cultures. Radicicol potently prevented the loss of neuronal cell bodies and neurites from LPS/IFN-gamma-induced neurotoxicity in rat cortical neuron-glia cultures with an EC(50) value of 0.09 microM. Radicicol inhibited the LPS/IFN-gamma-induced expression of inducible nitric oxide synthase (iNOS) and production of nitric oxide (NO) in microglia. Additionally, radicicol decreased the LPS/IFN-gamma-induced release of tumor necrosis factor-alpha (TNF-alpha) in the cultures. The inhibitory potency of radicicol against the production of NO and TNF-alpha was well correlated with the protection of neurons. These results suggest that the protective effect of radicicol against LPS/IFN-gamma-induced neuronal cell death in neuron-glia cultures is mediated via the inhibition of TNF-alpha release, as well as the suppression of iNOS expression in microglia.     

Longitudinal assessment of human immunodeficiency virus type 1 (HIV-1)-specific gamma interferon responses during the first year of life in HIV-1-infected infants

Human immunodeficiency virus type 1 (HIV-1) infection results in different patterns of viral replication in pediatric compared to adult populations. The role of early HIV-1-specific responses in viral control has not been well defined, because most studies of HIV-1-infected infants have been retrospective or cross-sectional. We evaluated the association between HIV-1-specific gamma interferon (IFN-gamma) release from the cells of infants of 1 to 3 months of age and peak viral loads and mortality in the first year of life among 61 Kenyan HIV-1-infected infants. At 1 month, responses were detected in 7/12 (58%) and 6/21 (29%) of infants infected in utero and peripartum, respectively (P = 0.09), and in approximately 50% of infants thereafter. Peaks of HIV-specific spot-forming units (SFU) increased significantly with age in all infants, from 251/10(6) peripheral blood mononuclear cells (PBMC) at 1 month of age to 501/10(6) PBMC at 12 months of age (P = 0.03), although when limited to infants who survived to 1 year, the increase in peak HIV-specific SFU was no longer significant (P = 0.18). Over the first year of life, infants with IFN-gamma responses at 1 month had peak plasma viral loads, rates of decline of viral load, and mortality risk similar to those of infants who lacked responses at 1 month. The strength and breadth of IFN-gamma responses at 1 month were not significantly associated with viral containment or mortality. These results suggest that, in contrast to HIV-1-infected adults, in whom strong cytotoxic T lymphocyte responses in primary infection are associated with reductions in viremia, HIV-1-infected neonates generate HIV-1-specific CD8+-T-cell responses early in life that are not clearly associated with improved clinical outcomes.