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Coronavirus disease 2019 (COVID-19) might impact disease progression in people living with HIV (PLWH), including those on effective combination antiretroviral therapy (cART). These individuals often experience chronic conditions characterized by proviral latency or low-level viral replication in CD4+ memory T cells and tissue macrophages. Pro-inflammatory cytokines, such as TNF-α, IL-1β, IL-6, and IFN-γ, can reactivate provirus expression in both primary cells and cell lines. These cytokines are often elevated in individuals infected with SARS-CoV-2, the virus causing COVID-19. However, it is still unknown whether SARS-CoV-2 can modulate HIV reactivation in infected cells. Here, we report that exposure of the chronically HIV-1-infected myeloid cell line U1 to two different SARS-CoV-2 viral isolates (ancestral and BA.5) reversed its latent state after 24 h. We also observed that SARS-CoV-2 exposure of human primary monocyte-derived macrophages (MDM) initially drove their polarization towards an M1 phenotype, which shifted towards M2 over time. This effect was associated with soluble factors released during the initial M1 polarization phase that reactivated HIV production in U1 cells, like MDM stimulated with the TLR agonist resiquimod. Our study suggests that SARS-CoV-2-induced systemic inflammation and interaction with macrophages could influence proviral HIV-1 latency in myeloid cells in PLWH.

HIV-1 remains a global health crisis 1 , highlighting the need to identify new targets for therapies. Here, given the disproportionate HIV-1 burden and marked human genome diversity in Africa 2 , we assessed the genetic determinants of control of set-point viral load in 3,879 people of African ancestries living with HIV-1 participating in the international collaboration for the genomics of HIV 3 . We identify a previously undescribed association signal on chromosome 1 where the peak variant associates with an approximately 0.3 log 10 -transformed copies per ml lower set-point viral load per minor allele copy and is specific to populations of African descent. The top associated variant is intergenic and lies between a long intergenic non-coding RNA (LINC00624) and the coding gene CHD1L, which encodes a helicase that is involved in DNA repair 4 . Infection assays in iPS cell-derived macrophages and other immortalized cell lines showed increased HIV-1 replication in CHD1L-knockdown and CHD1L-knockout cells. We provide evidence from population genetic studies that Africa-specific genetic variation near CHD1L associates with HIV replication in vivo. Although experimental studies suggest that CHD1L is able to limit HIV infection in some cell types in vitro, further investigation is required to understand the mechanisms underlying our observations, including any potential indirect effects of CHD1L on HIV spread in vivo that our cell-based assays cannot recapitulate., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

In addition to CD4 + T lymphocytes, myeloid cells and, particularly, differentiated macrophages are targets of human immunodeficiency virus type-1 (HIV-1) infection via the interaction of gp120Env with CD4 and CCR5 or CXCR4. Both T cells and macrophages support virus replication, although with substantial differences. In contrast to activated CD4 + T lymphocytes, HIV-1 replication in macrophages occurs in nondividing cells and it is characterized by the virtual absence of cytopathicity both in vitro and in vivo. These general features should be considered in evaluating the role of cell-associated restriction factors aiming at preventing or curtailing virus replication in macrophages and T cells, particularly in the context of designing strategies to tackle the viral reservoir in infected individuals receiving combination antiretroviral therapy. In this regard, we will here also discuss a model of reversible HIV-1 latency in primary human macrophages and the role of host factors determining the restriction or reactivation of virus replication in these cells.

In addition to CD4 + T cells, tissue-resident macrophages are target of productive HIV-1 infection. Unlike CD4 + T lymphocytes they are characterized by a substantial resistance to the cytopathic effects triggered by viral infection. This feature, in addition to their homeostatic self-renewal capacity, strongly support the hypothesis that macrophages could serve as an additional reservoir of persistently infected cells in individuals receiving combination antiretroviral therapy (cART).In order to study the peculiar aspects of HIV-1 infection of macrophages, human primary monocyte-derived macrophages (MDM) represent the most exploited model given the difficulty to obtain and maintain in culture for significant periods of time macrophages from different organs and tissues. Here we present a model of MDM differentiation achieved in the absence of addition of exogenous cytokines (such as GM-CSF, discussed in the previous chapter), that could be further investigated in term of cell polarization toward classic, proinflammatory "M1", or alternatively activated "M2" cells before or after infection. We will also discuss how to reinforce the M1-polarization protocol to obtain a reliable model of reversible latency of infectious HIV-1 in primary  M1-MDM., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

In the summer of 1981, a new deadly disease suddenly emerged targeting young men having sexwith men (MSM); three years later, a new virus, an exogenous human retrovirus, later named humanimmunodeficiency virus (HIV), was demonstrated to be the causative agent of the new disease, theAcquired Immuno-Deficiency Syndrome (AIDS), affecting, in addition to MSM, also intravenousdrug users, hemophiliacs, heterosexual individuals and children born to infected mothers. AIDSremained a dead sentence for >95% infected individuals until 1996 when the first combinationantiretroviral therapy (cART) was shown to be effective saving the lives of countless people. Sincethen, cART has become extremely powerful and simpler to adhere (now down to one or two pillsa day). However, virus eradication ("Cure") has been achieved thus far only in two individuals asa result of stem cell transplantation by an immunologically compatible donor homozygote for theCCR5Δ32 mutation; CCR5 is indeed the major entry coreceptor for the virus together with theprimary receptor CD4. This represents the exception to the rule that none of the many experimentalattempts of eliminating or silencing the virus reservoir unaffected by cART has achieved a significantproof of concept. In this article we will describe the essential aspects of the viral reservoirs and thecurrent strategies to tackle it.

As already discussed for T cell lines, also myeloid cell lines as served as the earliest models of chronic HIV infection. They were particularly relevant in the late 1980s and early 1990s when most experimental in vitro infections were based on laboratory-adapted "T-cell tropic" strains of HIV-1, such as LAI/IIIB or others, that later were found to rely upon CXCR4 as coreceptor for viral entry in addition to CD4 as primary receptor. Although primary macrophages do express CXCR4 together with CD4, virus replication is much less efficient than that observed with CCR5-using "macrophage-tropic" strains, as discussed separately in this book. Although different myeloid cell lines have been used to generate models of chronic HIV-1 infection that could be used to investigate features of proviral reactivation, as reviewed in (Cassol et al. J Leukoc Biol 80:1018-1030, 2006), two cell lines in particular have been broadly used and will be here discussed: the U937-derived U1 and HL-60-derived OM-10.1., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

The introduction of combination antiretroviral therapy (cART) has switched HIV-1 infection from a lethal disease to a chronic one. Indeed, cART is a lifelong treatment since its interruption is always followed by a rapid rebound of viremia from both cellular and anatomical viral reservoirs where the integrated HIV-1 provirus remains transcriptionally silent or maintains low-levels of viral replication, thereby preventing HIV-1 eradication. As therapeutic approach, the "shock and kill" strategy has emerged with the main objective to reactivate HIV-1 transcription from latency by using latency reversing agents (LRAs) prior to kill the reactivated infected cells by improving host immune responses. In this context, the development of tools such as HIV-1 latently infected cell lines have drastically increased our knowledge about HIV-1 latency and how to counteract this highly heterogeneous phenomenon. In this chapter, we will describe several chronically HIV-1 infected T-lymphocytic cell lines as useful surrogate models to study reversible HIV-1 proviral latency in CD4+ T cells in vitro before approaching more complex and expensive models., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Viral invasion of target cells triggers an immediate intracellular host defense system aimed at preventing further propagation of the virus. Viral genomes or early products of viral replication are sensed by a number of pattern recognition receptors, leading to the synthesis and production of type I interferons (IFNs) that, in turn, activate a cascade of IFN-stimulated genes (ISGs) with antiviral functions. Among these, several members of the tripartite motif (TRIM) family are antiviral executors. This article will focus, in particular, on TRIM22 as an example of a multitarget antiviral member of the TRIM family. The antiviral activities of TRIM22 against different DNA and RNA viruses, particularly human immunodeficiency virus type 1 (HIV-1) and influenza A virus (IAV), will be discussed. TRIM22 restriction of virus replication can involve either direct interaction of TRIM22 E3 ubiquitin ligase activity with viral proteins, or indirect protein-protein interactions resulting in control of viral gene transcription, but also epigenetic effects exerted at the chromatin level.

We investigated the contribution of human leukocyte antigen A2 (HLA-A2) and HLA-E-restricted CD8 + T cells in patients with Mycobacterium tuberculosis and human immunodeficiency virus 1 (HIV-1) coinfection. HIV-1 downregulates HLA-A, -B, and -C molecules in infected cells, thus influencing recognition by HLA class I-restricted CD8 + T cells but not by HLA-E-restricted CD8 + T cells, owing to the inability of the virus to downmodulate their expression. Therefore, antigen-specific HLA-E-restricted CD8 + T cells could play a protective role in Mycobacterium tuberculosis and HIV-1 coinfection. HLA-E- and HLA-A2-restricted Mycobacterium tuberculosis -specific CD8 + T cells were tested in vitro for cytotoxic and microbicidal activities, and their frequencies and phenotypes were evaluated ex vivo in patients with active tuberculosis and concomitant HIV-1 infection. HIV-1 and Mycobacterium tuberculosis coinfection caused downmodulation of HLA-A2 expression in human monocyte-derived macrophages associated with resistance to lysis by HLA-A2-restricted CD8 + T cells and failure to restrict the growth of intracellular Mycobacterium tuberculosis . Conversely, HLA-E surface expression and HLA-E-restricted cytolytic and microbicidal CD8 responses were not affected. HLA-E-restricted and Mycobacterium tuberculosis -specific CD8 + T cells were expanded in the circulation of patients with Mycobacterium tuberculosis /HIV-1 coinfection, as measured by tetramer staining, but displayed a terminally differentiated and exhausted phenotype that was rescued in vitro by anti-PD-1 (programmed cell death protein 1) monoclonal antibody. Together, these results indicate that HLA-E-restricted and Mycobacterium tuberculosis -specific CD8 + T cells in patients with Mycobacterium tuberculosis /HIV-1 coinfection have an exhausted phenotype and fail to expand in vitro in response to antigen stimulation, which can be restored by blocking the PD-1 pathway using the specific monoclonal antibody nivolumab.

HIV-1 infects CD4+ T lymphocytes with a 'helper' function and myeloid cells, mostly tissue-resident macrophages. While infection of CD4 T lymphocytes in the absence of combination antiretroviral therapy (cART) leads to their depletion and to a profound immunodeficiency, macrophages are resistant to virus-induced cytopathicity and are a source of infectious virus, particularly in the central nervous system (CNS). Infected macrophages are characterized by accumulating newly formed viral particles (virions) in subcellular vacuoles defined as 'virus-containing compartments (VCC)', derived from invaginations of the plasma membrane, that are poorly accessible to antiretroviral agents and anti-HIV antibodies. Several factors favor the accumulation of HIV-1 virions in VCC in vitro, whereas extracellular ATP, via binding to its receptor P2X7, is the only agent described thus far as capable of triggering the rapid release of VCC-sequestered virions without simultaneously causing the death of infected macrophages. Thus, the eATP/P2X7 axis could be exploited to achieve a pharmacological control of VCC-associated viral reservoir in individuals under effective cART., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

We have reported that short-term stimulation of primary human monocyte-derived macrophages (MDM) with interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), i.e. M1 polarization, leads to a significant containment of virus replication. Here we show that M1-MDM restimulation with these cytokines 7 days after infection (M1 2 MDM) promoted an increased restriction of HIV-1 replication characterized by very low levels of virus production near to undetectable levels. In comparison to control and M1-MDM that were not restimulated, M1 2 MDM showed a stronger reduction of both total and integrated HIV DNA as well as of viral mRNA expression. M1 2 MDM were characterized by an upregulated expression of restriction factors acting at the level of reverse transcription (RT), including apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3A (APOBEC3A) and APOBEC3G, but not SAM domain and HD domain-containing protein 1 (SAMHD1). M1 2 MDM also showed an increased expression of Class II Transactivator (CIITA) and Tripartite Motif22 (TRIM22), two negative regulators of proviral transcription, whereas expression and phosphorylation of transcriptional inducers of HIV-1, such as nuclear factor kB (NF-kB) and signal transducer and activator of transcription 1 (STAT1), were not impaired in these cells. The almost quiescent state of the infection in M1 2 MDM was promptly reversed by coculture with mitogen-stimulated leukocytes or cell incubation with their filtered culture supernatant. M1 2 MDM harbored replication-competent HIV-1 as virus spreading following cell stimulation was fully prevented by the RT inhibitor lamivudine/3TC. Selective reactivation of proviral expression in M1 2 MDM, but not in control or in M1-MDM that were not restimulated, was confirmed in cells infected with single round Vesicular Stomatitis Virus-G-pseudotyped HIV-1. Thus, M1 2 MDM represent an in vitro model of reversible, almost quiescent HIV-1 infection of primary human macrophages that could be further exploited for "Cure" related investigations.

HIV-1 insertions targeting BACH2 or MLK2 are enriched and persist for decades in hematopoietic cells from patients under combination antiretroviral therapy. However, it is unclear how these insertions provide such selective advantage to infected cell clones. Here, we show that in 30/87 (34%) patients under combination antiretroviral therapy, BACH2, and STAT5B are activated by insertions triggering the formation of mRNAs that contain viral sequences fused by splicing to their first protein-coding exon. These chimeric mRNAs, predicted to express full-length proteins, are enriched in T regulatory and T central memory cells, but not in other T lymphocyte subsets or monocytes. Overexpression of BACH2 or STAT5B in primary T regulatory cells increases their proliferation and survival without compromising their function. Hence, we provide evidence that HIV-1-mediated insertional activation of BACH2 and STAT5B favor the persistence of a viral reservoir in T regulatory cells in patients under combination antiretroviral therapy.HIV insertions in hematopoietic cells are enriched in BACH2 or MLK2 genes, but the selective advantages conferred are unknown. Here, the authors show that BACH2 and additionally STAT5B are activated by viral insertions, generating chimeric mRNAs specifically enriched in T regulatory cells favoring their persistence.

We investigated the potential anti-HIV-1 activity of the candidate microbicide 5-hydroxytyrosol (5-HT) both in primary human cervical tissue explants (CTE), established from tissues of women undergoing histerectomy, and in endometrium-associated leukocytes (EAL). CTE were exposed to either the laboratory-adapted HIV-1 BaL or to primary viral isolates in the presence or absence of 5-HT or 3TC/lamivudine as control and were then monitored for 12 days in terms of HIV-1 p24 Gag antigen production in culture supernatants. HIV-1 BaL replication was also evaluated in EAL by reverse transcriptase (RT) activity. The highest nontoxic concentrations of 5-HT (200 and 100 μM for CTE and EAL, respectively) exerted a significant inhibitory effect on virus replication in both primary cell systems. 5-HT did not cause significant alterations of the activation profile of CD4 + and CD8 + T cells, in terms of CD4, CCR5, CD25, CD69 and HLA-DR expression, although it decreased the percentage of CD38 + CD8 + T cells. Thus, 5-HT deserves consideration as a potential candidate microbicide for preventing HIV-1 transmission or curtailing its replication in the female reproductive tract., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Several studies demonstrated a relevant role of polymorphisms located within the HLA-B and -C loci and the Killer Immunoglobulin Receptors (KIRs) 3DL1 and 3DS1 in controlling HIV-1 replication. KIRs are regulatory receptors expressed at the surface of NK and CD8+ T-cells that specifically bind HLA-A and -B alleles belonging to the Bw4 supratype and all the -C alleles expressing the C1 or C2 supratype. We here disclose a novel signature associated with the Elite Controller but not with the long-term nonprogressor status concerning 2DS activating KIRs and HLA-C2 alleles insensitive to miRNA148a regulation. Overall, our findings support a crucial role of NK cells in the control of HIV-1 viremia.

In addition to CD4 T lymphocytes, HIV-1 infects tissue macrophages that can actively accumulate infectious virions in vacuolar subcellular structures mostly connected to the plasma membrane and recently termed virus-containing compartments (VCCs). The VCC-associated HIV-1 reservoir of infected macrophages can be either increased or depleted by immunologic and pharmacologic agents, at least in vitro, thus suggesting that these factors (or related molecules) could be effective in curtailing the macrophage-associated HIV-1 reservoir in infected individuals receiving combination antiretroviral therapy (cART). Here we review evidence on the pathogenic role of tissue macrophages as long-term viral reservoirs in vivo and upon in vitro infection with a particular emphasis on the immuno-pharmacological modulation of VCCs., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Introduction: Understanding the mechanisms by which some individuals are able to naturally control HIV-1 infection is an important goal of AIDS research. We here describe the case of an HIV-1(+) woman, CASE1, who has spontaneously controlled her viremia for the last 14 of her 20 years of infection., Methods: CASE1 has been clinically monitored since 1993. Detailed immunological, virological and histological analyses were performed on samples obtained between 2009 and 2011., Results: As for other Elite Controllers, CASE1 is characterized by low to undetectable levels of plasma HIV-1 RNA, peripheral blood mononuclear cell (PBMC) associated HIV-1 DNA and reduced in vitro susceptibility of target cells to HIV-1 infection. Furthermore, a slow rate of virus evolution was demonstrated in spite the lack of assumption of any antiretroviral agent. CASE1 failed to transmit HIV-1 to either her sexual male partner or to her child born by vaginal delivery. Normal values and ratios of T and B cells were observed, along with normal histology of the intestinal mucosa. Attempts to isolate HIV-1 from her PBMC and gut-derived cells were unsuccessful, despite expression of normal cell surface levels of CD4, CCRC5 and CXCR4. CASE1 did not produce detectable anti-HIV neutralizing antibodies in her serum or genital mucosal fluid although she displayed potent T cell responses against HIV-1 Gag and Nef. CASE1 also possessed multiple genetic polymorphisms, including HLA alleles (B*14, B*57, C*06 and C*08.02) and HLA-C single nucleotide polymorphisms (SNPs, rs9264942 C/C and rs67384697 del/del), that have been previously individually associated with spontaneous control of plasma viremia, maintenance of high CD4(+) T cell counts and delayed disease progression., Conclusions: CASE1 has controlled her HIV-1 viremia below the limit of detection in the absence of antiretroviral therapy for more than 14 years and has not shown any sign of immunologic deterioration or disease progression. Co-expression of multiple protective HLA alleles, HLA-C SNPs and strong T cell responses against HIV-1 proteins are the most likely explanation of this very benign case of spontaneous control of HIV-1 disease progression.

Cervical tissue explants (CTEs) from 22 HIV-1 seronegative women were exposed to R5 HIV-1 ex vivo. Eight CTEs were productively infected in terms of HIV-1 p24Gag release in culture supernatants, whereas 14 were not. Nonetheless, both accumulation of HIV-1gag DNA and of p24Gag(+) CD4(+) T cells and macrophages occurred in both productive and, at lower levels, in nonproductive CTEs. Nonproductive CTEs differed from productive CTEs for higher secretion of C-C motif chemokine ligand 3 (CCL3) and CCL5. A post-hoc analysis revealed that all productive CTEs were established from women in their secretory phase of the menstrual cycle, whereas nonproductive CTEs were derived from women either in their secretory (28%) or proliferative (36%) menstrual cycle phases or with an atrophic endometrium (36%). Thus, our results support the epidemiological observation that sexual HIV-1 transmission from males to women as well as from women to men is more efficient during their secretory phase of the menstrual cycle.

Background: Human immunodeficiency virus type 1 (HIV-1) infection is associated with a massive depletion of intestinal CD4(+) T cells that is only partially reversed by combination antiretroviral therapy (cART). Here, we assessed the ability of nucleoside reverse-transcriptase inhibitor/nonnucleoside reverse-transcriptase inhibitor treatment to restore the CD4(+) T-cell populations in the intestine of South African patients with AIDS., Methods: Thirty-eight patients with advanced HIV-1 infection who had chronic diarrhea (duration, >4 weeks) and/or unintentional weight loss (>10% decrease from baseline) of uncertain etiology were enrolled. Blood specimens were collected monthly, and gastrointestinal tract biopsy specimens were collected before cART initiation (from the duodenum, jejunum, ileum, and colon), 3 months after cART initiation (from the duodenum), and 6 months after cART initiation (from the duodenum and colon). CD4(+), CD8(+), and CD38(+)CD8(+) T cells were quantified by flow cytometry and immunohistochemistry analyses, and the HIV-1 RNA load was determined by the Nuclisens assay., Results: CD4(+) T-cell and HIV-1 RNA levels were significantly lower, whereas CD8(+) T-cell levels, including activated CD38(+)CD8(+) T cell levels, were higher in the duodenum and jejunum, compared with the colon. After 6 months of cART, a significant but incomplete recovery of CD4(+) T cells was detected in the colon and peripheral blood but not in the duodenum. Failed restoration of the CD4(+) T-cell count in the duodenum was associated with nonspecific enteritis and CD8(+) T-cell activation., Conclusions: Strategies that target inflammation and immune activation in the small intestine may be required to expedite CD4(+) T-cell recovery and improve therapeutic outcomes.

Objective(s): Tripartite motif-containing 22 (TRIM22) is an interferon-induced protein that inhibits HIV-1 transcription and replication in vitro. Two single nucleotide missense polymorphisms rs7935564A/G (SNP-1) and rs1063303C/G (SNP-2) characterize the coding sequence of human TRIM22 gene. We tested whether these variants affected the inhibitory effect of TRIM22 on HIV-1 replication and transcription and their potential association with HIV-1 disease., Design: The allelic discrimination was determined in 182 HIV-1-negative and among HIV-1-positive individuals with advanced disease progression (advanced progressors; n = 57), normal progressors (n = 76), and long-term nonprogressors (LTNPs; n = 95)., Methods: Renilla luciferase activity was measured after infection of activated peripheral blood mononuclear cells (PBMCs) from an additional group of 61 blood donors with a recombinant HIV-1. HIV-1-long terminal repeat (LTR)-driven luciferase activity was tested in the presence of plasmid expressing TRIM22 variants in 293T cells. The SNP genotyping was determined by TaqMan assay., Results: HIV-1 replication was more efficient in PBMCs from donors with SNP-1G and SNP-2G than from those with SNP-1A and SNP-2C alleles. Consistently, TRIM22-GG enhanced, whereas TRIM22-AC restricted basal HIV-1 LTR-driven transcription. In vivo, SNP-1G homozygotes and A/G heterozygotes were more frequent in advanced progressors than in LTNPs [odds ratio (OR) = 2.072, P = 0.005] or in normal progressors (OR = 1.809, P = 0.022); in contrast, SNP-2 was not associated with any state of HIV-1 disease progression. Although SNP-2 distribution was similar among the groups, TRIM22-GG haplotype was found more frequently in advanced progressors than in LTNPs (P = 0.02)., Conclusion: TRIM22 genetic diversity affects HIV-1 replication in vitro and it is a potentially novel determinant of HIV-1 disease severity., (© 2013 Wolters Kluwer Health | Lippincott Williams & Wilkins)

Cell-associated receptor for urokinase plasminogen activator (uPAR) is released as both full-length soluble uPAR (suPAR) and cleaved (c-suPAR) form that maintain ability to bind to integrins and other receptors, thus triggering and modulating cell signaling responses. Concerning HIV-1 infection, plasma levels of suPAR have been correlated with the severity of disease, levels of immune activation and ineffective immune recovery also in individuals receiving combination anti-retroviral therapy (cART). However, it is unknown whether and which suPAR forms might contribute to HIV-1 induced pathogenesis and to the related state of immune activation. In this regard, lymphoid organs represent an import site of chronic immune activation and virus persistence even in individuals receiving cART. Lymphoid organs of HIV-1(+) individuals showed an enhanced number of follicular dendritic cells, macrophages and endothelial cells expressing the cell-associated uPAR in comparison to those of uninfected individuals. In order to investigate the potential role of suPAR forms in HIV-1 infection of secondary lymphoid organs, tonsil histocultures were established from HIV-1 seronegative individuals and infected ex vivo with CCR5- and CXCR4-dependent HIV-1 strains. The levels of suPAR and c-suPAR were significantly increased in HIV-infected tonsil histocultures supernatants in comparison to autologous uninfected histocultures. Supernatants from infected and uninfected cultures before and after immunodepletion of suPAR forms were incubated with the chronically infected promonocytic U1 cell line characterized by a state of proviral latency in unstimulated conditions. In the contest of HIV-conditioned supernatants we established that c-suPAR, but not suPAR, inhibited chemotaxis and induced virus expression in U1 cells. In conclusion, lymphoid organs are an important site of production and release of both suPAR and c-suPAR, this latter form being endowed with the capacity of inhibiting chemotaxis and inducing HIV-1 expression.

Mononuclear phagocytes play a fundamental role in the tissue homeostasis and innate defenses against viruses and other microbial pathogens. In addition, they are likely involved in several steps of cancer development. Circulating monocytes and tissue macrophages are target cells of viral infections, including human cytomegalovirus, human herpes virus 8, and the HIV, and alterations of their functional and phenotypic properties are likely involved in many tissue-degenerative diseases, including atherosclerosis and cancer. Different tissue microenvironments as well as their pathological alterations can profoundly affect the polarization state of macrophages toward the extreme phenotypes conventionally termed M1 and M2. Thus, targeting disease-associated macrophages is considered a potential approach particularly in the context of cancer-associated tumor-associated macrophages, supporting malignant cell growth and progression toward a metastatic phenotype. Of note is the fact that tumor-associated macrophages isolated from established tumors display phenotypic and functional features similar to those of in vitro-derived M2-polarized cells. Concerning HIV-1 infection, viral eradication strategies in the context of combination antiretroviral therapy should also consider the possibility to deplete, at least transiently, certain mononuclear phagocytes subsets, although the possibility of distinguishing those that are either infected or pathogenically altered remains a goal of future research. In the present review, we will focus on the recent literature concerning the role of human macrophage polarization in viral infections and cancer.

The human immunodeficiency virus type-1 (HIV-1) is the etiological agent of the acquired immunodeficiency syndrome (AIDS), a disease highly lethal in the absence of combination antiretroviral therapy. HIV infects CD4(+) cells of the immune system (T cells, monocyte-macrophages and dendritic cells) via interaction with a universal primary receptor, the CD4 molecule, followed by a mandatory interaction with a second receptor (co-receptor) belonging to the chemokine receptor family. Apart from some rare cases, two chemokine receptors have been evolutionarily selected to accomplish this need for HIV-1: CCR5 and CXCR4. Yet, usage of these two receptors appears to be neither casual nor simply explained by their levels of cell surface expression. While CCR5 use is the universal rule at the start of every infection regardless of the transmission route (blood-related, sexual or mother to child), CXCR4 utilization emerges later in disease coinciding with the immunological deficient phase of infection. Moreover, in most instances CXCR4 use as viral entry co-receptor is associated with maintenance of CCR5 use. Since antiviral agents preventing CCR5 utilization by the virus are already in use, while others targeting either CCR5 or CXCR4 (or both) are under investigation, understanding the biological correlates of this "asymmetrical" utilization of HIV entry co-receptors bears relevance for the clinical choice of which therapeutics should be administered to infected individuals. We will here summarize the basic knowledge and the hypotheses underlying the puzzling and yet unequivocal role of CXCR4 in HIV-1 infection.

Multiple genome-wide association studies (GWAS) have been performed in HIV-1 infected individuals, identifying common genetic influences on viral control and disease course. Similarly, common genetic correlates of acquisition of HIV-1 after exposure have been interrogated using GWAS, although in generally small samples. Under the auspices of the International Collaboration for the Genomics of HIV, we have combined the genome-wide single nucleotide polymorphism (SNP) data collected by 25 cohorts, studies, or institutions on HIV-1 infected individuals and compared them to carefully matched population-level data sets (a list of all collaborators appears in Note S1 in Text S1). After imputation using the 1,000 Genomes Project reference panel, we tested approximately 8 million common DNA variants (SNPs and indels) for association with HIV-1 acquisition in 6,334 infected patients and 7,247 population samples of European ancestry. Initial association testing identified the SNP rs4418214, the C allele of which is known to tag the HLA-B*57:01 and B*27:05 alleles, as genome-wide significant (p = 3.6 × 10⁻¹¹). However, restricting analysis to individuals with a known date of seroconversion suggested that this association was due to the frailty bias in studies of lethal diseases. Further analyses including testing recessive genetic models, testing for bulk effects of non-genome-wide significant variants, stratifying by sexual or parenteral transmission risk and testing previously reported associations showed no evidence for genetic influence on HIV-1 acquisition (with the exception of CCR5Δ32 homozygosity). Thus, these data suggest that genetic influences on HIV acquisition are either rare or have smaller effects than can be detected by this sample size.

We performed a genome-wide association study comparing a cohort of 144 human immunodeficiency virus (HIV type 1-infected, untreated white long-term nonprogressors (LTNPs) with a cohort of 605 HIV-1-infected white seroconverters. Forty-seven single-nucleotide polymorphisms (SNPs), located from class I to class III major histocompatibility complex (MHC) subregions, show statistical association (false discovery rate, <0.05) with the LTNP condition, among which 5 reached genome-wide significance after Bonferonni correction. The MHC LTNP-associated SNPs are ordered in ≥4 linkage disequilibrium blocks; interestingly, an MHC class III linkage disequilibrium block (defined by the rs9368699 SNP) seems specific to the LTNP phenotype.

Delineation of the immune correlates of protection in natural infection or after vaccination is a mandatory step for vaccine development. Although the most recent techniques allow a sensitive and specific detection of the cellular immune response, a consensus on the best strategy to assess their magnitude and breadth is yet to be reached. Within the AIDS Vaccine Integrated Project (AVIP http://www.avip-eu.org) we developed an antigen scanning strategy combining the empirical-based approach of overlapping peptides with a vast array of database information. This new system, termed Variable Overlapping Peptide Scanning Design (VOPSD), was used for preparing two peptide sets encompassing the candidate HIV-1 vaccine antigens Tat and Nef. Validation of the VOPSD strategy was obtained by direct comparison with 15mer or 20mer peptide sets in a trial involving six laboratories of the AVIP consortium. Cross-reactive background responses were measured in 80 HIV seronegative donors (HIV-), while sensitivity and magnitude of Tat and Nef-specific T-cell responses were assessed on 90 HIV+ individuals. In HIV-, VOPSD peptides generated background responses comparable with those of the standard sets. In HIV-1+ individuals the VOPSD pools showed a higher sensitivity in detecting individual responses (Tat VOPSD vs. Tat 15mers or 20mers: p≤0.01) as well as in generating stronger responses (Nef VOPSD vs. Nef 20mers: p<0.001) than standard sets, enhancing both CD4 and CD8 T-cell responses. Moreover, this peptide design allowed a marked reduction of the peptides number, representing a powerful tool for investigating novel HIV-1 candidate vaccine antigens in cohorts of HIV-seronegative and seropositive individuals., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The Bridging Sheet domain of HIV-1 gp120 is highly conserved among the HIV-1 strains and allows HIV-1 binding to host cells via the HIV-1 coreceptors. Further, the bridging sheet domain is a major target to neutralize HIV-1 infection. We rationally designed four linear peptide epitopes that mimic the three-dimensional structure of bridging sheet by using molecular modeling. Chemically synthesized peptides BS3 and BS4 showed a fair degree of antigenicity when tested in ELISA with IgG purified from HIV(+) broadly neutralizing sera while the production of synthetic peptides BS1 and BS2 failed due to their high degree of hydrophobicity. To overcome this limitation, we linked all four BS peptides to the COOH-terminus of GST protein to test both their antigenicity and immunogenicity. Only the BS1 peptide showed good antigenicity; however, no envelope specific antibodies were elicited upon mice immunization. Therefore we performed further analyses by linking BS1 peptide to the NH2-terminus of the E2 scaffold from the Geobacillus Stearothermophylus PDH complex. The E2-BS1 fusion peptide showed good antigenic results, however only one immunized rabbit elicited good antibody titers towards both the monomeric and oligomeric viral envelope glycoprotein (Env). In addition, moderate neutralizing antibodies response was elicited against two HIV-1 clade B and one clade C primary isolates. These preliminary data validate the peptide mimotope approach as a promising tool to obtain an effective HIV-1 vaccine.

Background: Microbial translocation contributes to immune activation and disease progression during chronic human immunodeficiency virus type 1 (HIV-1) infection. However, its role in the African AIDS epidemic remains controversial. Here, we investigated the relationship between markers of monocyte activation, plasma lipopolysaccharide (LPS), and HIV-1 RNA in South Africans prioritized to receive combination antiretroviral therapy (cART)., Methods: Ten HIV-1-negative African controls and 80 HIV-1-infected patients with CD4 T cell counts <200 cells/microL were sampled prior to (n=60) or during (n=20) receipt of effective cART. Viral load was measured by Nuclisens; LPS by the Limulus amoebocyte lysate assay; monocyte and T cell subsets by flow cytometry; and soluble CD14, cytokines, and chemokines by enzyme-linked immunosorbent assay and customized Bio-Plex plates., Results: Three distinct sets of markers were identified. CCL2, CXCL10, and CD14(+)CD16(+) monocyte levels were positively correlated with HIV-1 viremia. This finding, together with cART-induced normalization of these markers, suggests that their upregulation was driven by HIV-1. Plasma interleukin-6 was associated with the presence of opportunistic coinfections. Soluble CD14 and tumor necrosis factor were linked to plasma LPS levels and, as observed for LPS, remained elevated in patients receiving effective cART., Conclusions: Microbial translocation is a major force driving chronic inflammation in HIV-infected Africans receiving cART. Prevention of monocyte activation may be especially effective at enhancing therapeutic outcomes.

In 1995, a breakthrough paper showed that intermittent cycles of interleukin-2 (IL-2), together with suboptimal ART, caused an unprecedented, stable increase in CD4+ T cell counts, without altering the steady state levels of viremia. At the time, this was somewhat obscured by the first successes of combination antiretroviral therapy (cART). However, since then, numerous studies have confirmed this basic finding, opening up a new perspective in the long-term management of chronic HIV infection. One of the benchmarks of this experimental treatment is the expansion of CD4+CD25+ T lymphocytes probably including T regulatory cells (Tregs). Based on these encouraging findings, two major phase III clinical trials, ESPRIT and SILCAAT, involving thousands of patients worldwide, were launched and continued over several years. Unfortunately, they both resulted in the highly unexpected, yet unequivocal, outcome of a lack of a protective effect of IL-2-expanded CD4+ T cells on HIV disease progression towards the acquired immunodeficiency syndrome (AIDS) or death. In addition, there was the suggestion of an increase in certain deleterious effects on treated patients in terms of cardiovascular and inflammatory events. While IL-2 therapy is unlikely to be studied any further in the context of HIV infection, other cytokines, such as IL-7, are still being tested in the hope of more promising results.

The genetic diversity of HIV-1 strains circulating among HIV-1-infected South Africans was investigated in a cohort of 420 individuals enrolled as part of the AIDS Vaccine Integrated Project (AVIP) study. Representative samples (10%) were randomly selected from treatment-naive participants. Viral RNA was extracted for reverse transcriptase-initiated amplification and population-based sequencing of partial pol (encompassing protease and reverse transcriptase) and full-length integrase. Overall, HIV-1 sequences confirmed that 97.1% and 96.9% were HIV-1 subtype C in pol and integrase, respectively. Two participants were infected with unique A1/C and C/A1 recombinants in pol/integrase. Further pol sequence analysis identified mutation patterns associated with high level resistance to NNRTIs in two participants, whereas no primary mutations conferring resistance to integrase inhibitors were detected. The predominance of HIV-1 subtype C in South African populations is therefore confirmed in the AVIP cohort finalized for testing preventive or therapeutic vaccines against HIV-1 infection.

Objective: To investigate the impact of intermittent interleukin-2 (IL-2) plus combination antiretroviral therapy (cART) on HIV-1 entry co-receptor use., Methods: Primary HIV-1 isolates were obtained from 54 HIV-1-positive individuals at baseline and after 12 months using co-cultivation of peripheral blood mononuclear cells (PBMC) with activated PBMC of HIV-negative healthy donors. HIV-1 co-receptor use was determined on U87-CD4 cells., Results: Fourteen out of the 21 (67%) IL-2-treated individuals harbouring a primary CCR5-dependent (R5) HIV-1 isolate at baseline confirmed an R5 virus isolation after 12 months in contrast to 3 out of 7 (43%) of those receiving cART only. After 12 months, only 1 R5X4 HIV-1 isolate was obtained from 21 cART+IL-2-treated individuals infected with an R5 virus at entry (5%) vs. 2/7 (29%) patients receiving cART alone, as confirmed by a 5-year follow-up on some individuals., Conclusions: Intermittent IL-2 administration plus cART may prevent evolution towards CXCR4 usage in individuals infected with R5 HIV-1.

Polarization of MP into classically activated (M1) and alternatively activated (M2a, M2b, and M2c) macrophages is critical in mediating an effective immune response against invading pathogens. However, several pathogens use these activation pathways to facilitate dissemination and pathogenesis. Viruses generally induce an M1-like phenotype during the acute phase of infection. In addition to promoting the development of Th1 responses and IFN production, M1 macrophages often produce cytokines that drive viral replication and tissue damage. As shown for HIV-1, polarization can also alter macrophage susceptibility to infection. In vitro polarization into M1 cells prevents HIV-1 infection, and M2a polarization inhibits viral replication at a post-integration level. M2a cells also express high levels of C-type lectins that can facilitate macrophage-mediated transmission of HIV-1 to CD4(+) T cells. Macrophages are particularly abundant in mucosal membranes and unlike DCs, do not usually migrate to distal tissues. As a result, macrophages are likely to contribute to HIV-1 pathogenesis in mucosal rather than lymphatic tissues. In vivo polarization of MP is likely to span a spectrum of activation phenotypes that may change the permissivity to and alter the outcome of HIV-1 and other viral infections.

Objective: High mobility group box-1 (HMGB1) is a nuclear chromatin protein. Furthermore, it induces chemotaxis and inflammation once released in the extracellular milieu, and it has been reported to upregulate, but also to inhibit HIV-1 replication in different cell types. We here investigated the potential role of extracellular HMGB1 in both R5 and X4 HIV-1 replication in primary human monocyte-derived macrophages (MDM) and U937 promonocytic cells, respectively., Design: MDM or U937 cells were infected with R5 and X4 HIV-1 strains, respectively, in the presence or absence of endotoxin-free recombinant (r) HMGB1 or necrotic cell supernatants either containing or depleted of endogenous HMGB1., Methods: HIV replication was measured by means of virion-associated reverse transcriptase activity in culture supernatants and cell-associated viral protein expression. Cytokine and chemokine production were measured by enzyme-linked immunosorbent assay; cell surface expression of CD4, CC chemokine receptor 5, receptor for advanced glycation end-products, Toll-like receptor-2 and Toll-like receptor-4 were analyzed by flow cytometry., Results: Both rHMGB1 and necrotic cell supernatant-associated HMGB1 inhibited replication of R5 HIV-1 in MDM. Surprisingly enough, no upregulation of CC chemokine receptor 5-binding chemokines or of other chemokines and cytokines was observed in rHMGB1-stimulated MDM. HMGB1 also induced chemotaxis and strongly inhibited the replication of X4 HIV-1 in the 'Minus' subset of U937 cell clones expressing high levels of putative HMGB1 receptors (receptor for advanced glycation end-products, Toll-like receptors 2 and 4)., Conclusion: Extracellular HMGB1 is a potent inhibitor of both R5 and X4 HIV-1 replication in mononuclear phagocytic cells without inducing the release of HIV-Modulatory chemokines or cytokines.

Urokinase-type plasminogen activator (uPA) signaling via its receptor uPAR inhibits late events in HIV-1 replication in acutely infected primary monocyte-derived macrophages (MDMs) and promonocytic U937 cells. Here we show that U937-derived, chronically infected U1 cells stimulated with phorbol 12-myristate 13-acetate (PMA) express integrins, uPA, and soluble uPAR at levels similar to those of MDMs. uPA inhibited HIV expression in U1 cells incubated with either PMA or tumor necrosis factor-alpha (TNF-alpha), but not with other HIV-inductive cytokines or lipopolysaccharide. Of interest, only PMA and TNF-alpha, but not other HIV-inductive stimuli, induced surface expression of the alpha(M) chain CD11b in U1 cells constitutively expressing CD18, the beta(2) chain of the Mac-1 integrin. Like uPA, fibrinogen, a Mac-1 (CD11b/CD18) ligand, and M25, a peptide homologous to a portion of the beta-propeller region of CD11b preventing its association with uPAR, inhibited HIV virion release in PMA-stimulated U1 cells. Both uPAR small-interference RNA (siRNA) and soluble anti-beta(1)/-beta(2) monoclonal antibodies abolished the anti-HIV effects of uPA, whereas CD11b siRNA reversed the anti-HIV effect of M25, but not that induced by uPA. Thus, either uPA/uPAR interaction, Mac-1 activation, or prevention of its association with uPAR triggers a signaling pathway leading to the inefficient release of HIV from monocytic cells.

INITIO is an open-labelled randomized trial evaluating first-line therapeutic strategies for human immunodeficiency virus-1 (HIV-1) infection. In an immunology substudy a tetanus toxoid booster (TTB) immunization was planned for 24 weeks after initiation of highly active antiretroviral therapy (HAART). All patients had received tetanus toxoid immunization in childhood. Generation of proliferative responses to tetanus toxoid was compared in two groups of patients, those receiving a protease inhibitor (PI)-sparing regimen (n = 21) and those receiving a PI-containing (n = 54) regimen. Fifty-two participants received a TTB immunization [PI-sparing (n = 15), PI-containing (n = 37)] and 23 participants did not [PI-sparing (n = 6) or PI-containing (n = 17)]. Cellular responses to tetanus antigen were monitored by lymphoproliferation at time of immunization and every 24 weeks to week 156. Proportions with a positive response (defined as stimulation index > or = 3 and Delta counts per minute > or = 3000) were compared at weeks 96 and 156. All analyses were intent-to-treat. Fifty-two participants had a TTB immunization at median 25 weeks; 23 patients did not. At weeks 96 and 156 there was no evidence of a difference in tetanus-specific responses, between those with or without TTB immunization (P = 0.2, P = 0.4). There was no difference in the proportion with response between those with PI-sparing or PI-containing regimens at both time-points (P = 0.8, P = 0.7). The proliferative response to tetanus toxoid was unaffected by initial HAART regimen. Anti-tetanus responses appear to reconstitute eventually in most patients over 156 weeks when treated successfully with HAART, irrespective of whether or not a TTB immunization has been administered.

Cytokines are involved early in the pathogenesis of HIV infection and disease progression as a component of immunologic dysregulation and immunodeficiency and as determinants controlling virus replication. Several steps, before and after retroviral integration into host DNA in T cells and macrophages, are affected by cytokines whereas CCR5 and CXCR4 binding chemokines can interfere with viral entry. A growing number of potential players--including the gamma-common interleukin (IL)-7, IL-15, and IL-21 together with IL-17, IL-18, IL-19, IL-20, IL-23, and IL-27--are discussed in terms of their perturbation in HIV infection and of their effects on virus replication. Thus, an increasing intersection of HIV infection and the cytokine network represents a crucial determinant of virus replication and immunologic dysregulation and will likely play a key role in the development of effective strategies of HIV prevention and immunologic reconstitution.

Urokinase-type plasminogen activator (uPA), an inducer of macrophage adhesion, inhibits HIV-1 expression in PMA-stimulated, chronically infected U1 cells. We investigated whether uPA-dependent cell adhesion played a role in uPA-dependent inhibition of HIV-1 replication in these cells. Monocyte-derived macrophages (MDM) were generated from monocytes of HIV-infected individuals or from cells of seronegative donors infected acutely in vitro. U1 cells were stimulated in the presence or absence of uPA in standard tissue culture (TC) plates, allowing firm cell adhesion or ultra-low adhesion (ULA) plates. Moreover, U1 cells were also maintained in the presence or absence of vitronectin (VN)-containing sera or serum from VN(-/-) mice. Virus production was evaluated by RT activity in culture supernatants, whereas cell adhesion was by crystal violet staining and optical microscopy. uPA inhibited HIV replication in MDM and PMA-stimulated U1 cells in TC plates but not in ULA plates. uPA failed to inhibit HIV expression in U1 cells stimulated with IL-6, which induces virus expression but not cell adhesion in TC plates. VN, known to bind to the uPA/uPA receptor complex, was crucial for these adhesion-dependent, inhibitory effects of uPA on HIV expression, in that they were not observed in TC plates in the presence of VN(-/-) mouse serum. HIV production in control cell cultures was increased significantly in ULA versus TC plates, indicating that macrophage cell adhesion per se curtails HIV replication. In conclusion, uPA inhibits HIV-1 replication in macrophages via up-regulation of cell adhesion to the substrate mediated by VN.

Background and Objective: Hepatitis C virus (HCV) infection is frequent in HIV-positive subjects. We evaluated the potential impact of HCV coinfection and other determinants on HIV disease progression in a cohort of long-term non-progressors (LTNPs)., Study Design: We studied immunological and virological factors in a cohort of 49 LTNPs, 23 of whom progressed during the follow-up (late progressors; LPs)., Results: HCV coinfection was detected in 19/26 LTNPs and 15/23 LPs. Univariate analysis showed that HIV viral load was associated with disease progression (P=0.04), and time-to-event analysis indicated that HCV genotype 1 significantly correlated with LTNP status (P=0.031). At multivariate analysis, HIV viremia at study entry remained independently associated with LTNP status (P=0.049). When the most represented genotypes (1 and 3a) were considered in the model, genotype 3a infection (P=0.034) and gender (P=0.035) emerged as independent variables related to HIV disease progression, whereas HIV viral load disappeared., Conclusions: In addition to HIV viremia, coinfection with different HCV genotypes and gender may affect LTNP status.

Background: The long-term immunological benefit of protease inhibitor (PI)-sparing antiretroviral therapy (ART) using non-nucleoside reverse transcriptase inhibitors (NNRTIs) remains poorly investigated., Methods: A total of 120 ART-naive, HIV-1-infected participants were included in the immunology substudy of INITIO, an international randomized trial comparing two NRTIs (didanosine + stavudine) combined with either: one NNRTI (efavirenz; EFV), one non-boosted PI (nelfinavir; NFV), or one NNRTI + one PI (EFV/NFV). CD4+ T-cell counts, HIV-1 plasma RNA load (VL), T-cell phenotype, T-cell proliferation and IFN-gamma production against opportunistic/recall and HIV-1 antigens/peptides were compared at baseline and at week (W) 96 and W156., Results: Participants (37 EFV, 44 NFV, 39 EFV/NFV) had similar baseline VL; median CD4+ T-cell counts/mm3 were: 144 (64-303) EFV, 212 (42-313) NFV and 257 (86-331) EFV/NFV. At W156, the proportion of patients with VL < or =50 copies/ml was not different between the arms (P=0.3). From baseline to W156 there was a significant increase in CD4+ T-cell counts (P<0.001) and in naive CD4+ T cells (P<0.001), with no difference between arms and percentages of total and activated CD8+ T cells decreased significantly (P<0.001) in all arms. The decrease in activated memory CD4+ T-cells was significantly greater in the EFV arm at W96 (P=0.03) and W156 (P=0.01), but did not persist after adjusting for baseline CD4+ T-cell counts. During follow-up, responses to opportunistic pathogens increased in all patients while specific T-cell responses to HIV-1-p24 and gp160 recombinant proteins or to Gag and Nef peptides were not restored., Conclusion: Regimens using/sparing PIs provide similar levels of long-term immune reconstitution even in patients with low CD4+ T-cell counts.

Infection with human immunodeficiency virus (HIV)-encoding defective nef variants may contribute to a relatively benign course of disease in a minority of long-term nonprogressors (LTNP). We have examined the functions of nef alleles from six individuals belonging to the same cohort of hemophiliacs infected with HIV-1 prior to 1985 and classified as LTNP in 1995. Three out of six individuals have progressed to HIV disease (late progressors [LP]), whereas the three remainders have maintained their LTNP status at least up to 2003. The nef alleles were obtained from both plasma virus and peripheral blood mononuclear cells of all six individuals in 1995 and 1998. The proportion of sequences containing mutations not yielding Nef expression significantly diminished in 1998 versus that in 1995. Several previously defined functional regions of intact nef alleles were highly conserved. However, the major variant obtained in 1998 from plasma RNA of five out of six individuals significantly reduced HIV infectivity/replication and impaired Nef-mediated CD4 but not major histocompatibility complex class I antigen down-modulation from the cell surface. Thus, functional alterations of the nef gene are present in both LP and LTNP, suggesting that Nef defectiveness in vitro is not necessarily associated with the long-term maintenance of LTNP status. Of interest is the fact that isolates from three out of three LP showed a dual CCR5/CXCR4 coreceptor use (R5X4), in contrast to those from LTNP, which were exclusively R5. Thus, in vivo evolution of gp120 Env to CXCR4 use appears to be associated with HIV disease progression in individuals infected with nef-defective viruses.

HIV infection of mononuclear phagocytes (MP), mostly as tissue macrophages, is a dominant feature in the pathogenesis of HIV disease and its progression to AIDS. Although the general mechanism of infection is not dissimilar to that of CD4+ T lymphocytes occurring via interaction of the viral envelope with CD4 and a chemokine receptor (usually CCR5), other features are peculiar to MP infection. Among others, the long-term persistence of productive infection, sustained by the absence of substantial cell death, and the capacity of the virions to bud and accumulate in intracellular multivesicular bodies (MVB), has conferred to MP the role of "Trojan horses" perpetuating the chronic state of infection. Because the investigation of tissue macrophages is often very difficult for both ethical and practical reasons of accessibility, most studies of in vitro infection rely upon monocyte-derived macrophages (MDM), a methodology hampered by inter-patient variability and lack of uniformity of experimental protocols. A number of cell lines, mostly Mono Mac, THP-1, U937, HL-60, and their derivative chronically infected counterparts (such as U1 and OM-10.1 cell lines) have complemented the MDM system of infection providing useful information on the features of HIV replication in MP. This article describes and compares the most salient features of these different cellular models of MP infection by HIV.

Before performing phase II/III clinical trials in Africa, preliminary studies, including assessment and building up of clinical and laboratory infrastructures, estimates of human immunodeficiency virus incidence, investigation of the background immune response, and evaluation of the cross-clade immune response, need to be done. Plans and ongoing work in the context of the AIDS Vaccine Integrated Project and some preliminary data are presented.

Bordetella pertussis toxin B-oligomer (PTX-B) has been shown to inhibit HIV infection and replication in vitro. The potential anti-viral effect of PTX-B was tested here in an in vivo surrogate model of HIV infection, i.e. SCID mice reconstituted with human peripheral blood leukocytes (PBL) (hu-PBL-SCID) and infected with a CCR5-dependent (R5) HIV-1 strain. SCID mice inoculated intra-peritoneal (i.p.) with PTX-B and then infected with the R5 strain SF-162 were sacrificed 7 days later and analyzed for human PBL (hu-PBL) lymphoid tissue reconstitution, infection of hu-PBL, plasma viremia and viral rescue from ex vivo-cultivated i.p. hu-PBL. Unlike mice treated with 500 ng per animal of PTX-B showing no evidence of viral inhibition, daily administration of PTX-B (50 ng per mouse) strongly inhibited virus infection and replication, as determined by undetectable viremia, absence of infected hu-PBL and lack of rescue of infectious HIV in most animals. Furthermore, PTX-B injection 2 h before and twice after infection prevented HIV-1 infection and replication in all (10/10) tested animals. Thus, PTX-B potently inhibited virus infection and replication in hu-PBL-SCID mice, supporting the hypothesis that it may represent a new pharmacological agent against HIV-1 infection.

The earliest defense against microbial infection is represented by the responses of the innate (or natural) immune system, that also profoundly regulates the adaptive (or acquired) T- and B-cell immune responses. Activation of the innate immune system is primed by microbial invasion in response to conserved structures present in large groups of microorganisms (LPS, peptidoglycan, double-stranded RNA), and is finely tuned by different cell types (including dendritic cells, macrophages, natural killer cells, natural killer T cells, and gammadelta T cells). In addition, several soluble factors (complement components, defensins, mannose-binding lectins, interferons, cytokines and chemokines) can play a major role in the regulation of both the innate and adaptive immunity. In this review, we will briefly overview the regulation of some cellular subsets of the innate immune system particularly involved in human immunodeficiency virus (HIV) infection and then focus our attention on those cytokines and chemokines whose levels of expression are more profoundly affected by HIV infection and that, conversely, can modulate virus infection and replication.

Natural toxins are the product of a long-term evolution, and have captured crucial events in the most essential and vital processes of living organisms. They can attack components of the protein synthesis machinery (as in the case of Diphteria and Shiga toxins, and Ribosome inactivating proteins), actin polymerization (Clostridium botulinum type C, C2, toxins and Enterotoxin A), signal transduction pathways (Cholera toxin, Heat-labile enterotoxins, Pertussis and Adenylate cyclase toxins), intracellular trafficking of vesicules (for Tetanus and Botulinum neurotoxin type C) as well as immune and/or inflammatory responses (Pyrogenic exotoxins, Cholera and Pertussis toxins). Of interest is the fact that several bacterial and vegetal toxins can either kill selectively cells infected with the human immunodeficiency virus (HIV) or exert inhibitory effects on its life cycle. In particular both pertussis toxin (PTX) and its nontoxic B-oligomeric component (PTX-B) can block the infectious process in vitro at multiple levels, by preventing the entry of CCR5-dependent (R5) HIV strains and by inhibiting both R5 and CXCR4-dependent HIVs at post-entry level(s). In addition, some toxins possess immunostimulating properties that have been exploited in terms of adjuvancy and induction of specific cytotoxic T lymphocytes responses to different vaccine preparations, including some experimental vaccine against HIV infection. Thus, toxins may represent a relatively unexplored exhibition of powerful biological agents that could either prevent infection or attack HIV-infected cells.

Dendritic cells (DC) and natural killer (NK) cells, the main cellular components of the innate immune system, participate in the most ancient first line of defense against infections. Both types of cells patrol peripheral tissues, whereas their rapid recruitment and activation at mucosal surfaces [the major entry point for the human immunodeficiency virus (HIV)] is a hallmark of acute inflammatory response. The ability of HIV to survive and replicate in the human host relies upon several molecular mechanisms eluding the immune surveillance of both adaptive immunity and of DC and NK cells beginning with the acute phase of primary HIV infection. DC and NK cells, unlike CD4+ T cells, are impaired more functionally rather than being depleted by HIV infection. In this article we will review some of the aspects of DC/NK cells interaction with HIV infection both in vitro and in vivo, and we will also speculate on the potential consequence for HIV pathogenesis and for the capacity of the virus to escape the surveillance of the innate immune system.

Background: Interleukin-2 (IL-2) has been investigated as an adjunct to antiretroviral therapy (ART) because of its well-demonstrated capacity of stably increasing the number of peripheral CD4+ T cell lymphocytes. However, IL-2-related adverse events (AEs), including fever, tachycardia, hypotension, and respiratory failure, are typically dose- and schedule-dependent and can potentially limit the application of IL-2 therapy in an outpatient setting. Nitric oxide (NO) is a potent vasodilator potentially responsible for some of the AEs caused by IL-2., Purpose: In this study, we determined NO production in a cohort of HIV-1 infected individuals receiving ART either alone or together with IL-2., Method: NO production, detected as plasma nitrate/nitrite levels by the Griess reaction, was evaluated in 3 groups of 10 individuals each. In the first group, subcutaneous (sc) administration of 12-15 million international units per day (MIU/d) of IL-2 was administered for 5 days every 8 weeks for 6 cycles together with ART; in the second group, IL-2 (6 MIU/d) was given sc for 5 days every 4 weeks for 12 cycles together with ART; whereas the third group received ART alone., Results: At baseline, the plasma nitrate/nitrite levels in the 2 groups of patients who received high and low doses of the cytokine along with ART were 28.5 +/- 18.1 micromol/L and 34.2 +/- 29.0 micromol/L, respectively. These levels were comparable to those of patients treated with only ART (18.6 +/- 22.4 micromol/L) and to those of 20 healthy controls (19.9 +/- 5.9 micromol/L). No significant increase of plasma nitrate/nitrite levels was observed by administration of either ART or ART+IL-2. In addition, NO production was not associated significantly with different levels of tumor necrosis factor-alpha, IL-6, or soluble IL-2 receptor alpha chain in 9 individuals with WHO grade 2 and 3 AEs., Conclusion: Our results indicate that NO is unlikely to be responsible for most side effects of IL-2 therapy in HIV-1 infected individuals. Because both IL-2 and virus multiplication have been reported to independently stimulate NO production, concomitant ART may curtail NO production through inhibition of HIV-1 replication.

The ideal microbicide must fulfill a number of criteria including a broad and potent activity against transmission of HIV and other sexually transmitted agents in the absence of toxicity and inflammation. We have described that derivatives of K5 polysaccharide from Escherichia coli inhibit HIV entry in target cells. K5 derivatives have a structure that resembles that of heparin, but they are devoid of the anticoagulant activity typical of heparin. Moreover, in contrast to heparin, they inhibit a broad spectrum of HIV-1 laboratory-adapted and primary isolates that use either CCR5 or CXCR4 or both coreceptors in terms of their infection and replication in primary CD4+ lymphocytes and monocytes-derived macrophages (MDM). Therefore, these compounds could be developed as candidate microbicides for preventing sexual HIV transmission, a predominant modality of HIV spreading in both the developed and underdeveloped world.