Category Archives: 3. Pathogenesis of HIV-1 Infection

Pathogenesis of HIV-1 Infection

(Andrea Rubbert, Georg Behrens and Mario Ostrowski)

Since the initial description of the Human Immunodeficiency Virus type I (HIV-1) in 1983 (Barré-Sinoussi 1983, Gallo 1983) and HIV-2 in 1986 (Clavel 1986), these two viruses have been identified as the primary cause of Acquired Immunodeficiency Syndrome (AIDS). As HIV-1 is the major cause of AIDS in the world today, our discussion will be primarily limited to HIV-1 infection. Worldwide, the number of HIV-1 infected persons exceeds 33 millions (according to UNAIDS), the majority of whom live in the developing countries of Sub-Saharan Africa, Asia and South America.

Despite all the therapeutic advantages achieved over the last decade, including the evolution of “HAART”, once an individual has become infected, eradication of the virus is not possible. In addition, new problems relating to the short- and long-term toxicity of drug treatments and the occurrence of resistance mutations in both circulating and transmitted viruses are emerging. In most countries in South East Asia and Africa, the incidence and prevalence of HIV-1 infection continues to increase and surpass that of Europe and North America.

However, due to the high costs of drug regimens and the lack of healthcare infrastructure in these developing countries, the widespread use of ART is currently still partial at best. The further course of the HIV-1 pandemic, therefore, mainly depends on how and to what degree developing countries with a high HIV-1 prevalence are able to take advantage of the medical progress achieved in Europe and North America, and whether an effective prophylactic vaccine will become available in the near future (see also chapter on Preventive HIV-1-Vaccine).

An understanding of the immunopathogenesis of HIV-1 infection is a major prerequisite for rationally improving therapeutic strategies, developing immunotherapeutics and prophylactic vaccines. As in other virus infections, the individual course of HIV-1 infection depends on both host and viral factors.

The course of infection with HIV-1 in HIV-infected humans may vary dramatically, even if the primary infection comes from the same source (Liu 1997). In individuals with a long-term non-progressive HIV-1 infection (i.e., lack of decline in CD4 T cell counts, or chronic infection for at least 7 years without the development of AIDS), a defective virion has been identified (Kirchhoff 1995). Thus, infection with a defective virus, or one that has a poor capacity to replicate, may prolong the clinical course of HIV-1 infection. However, in most individuals, HIV-1 infection is characterized by a replication-competent virus with a high daily turnover of virions.

Host factors may also determine whether or not an HIV-1-infected individual rapidly develops clinically overt immunodeficiency, or whether this individual belongs to the group of long-term non-progressors that represent about 5% of all infected patients. The identification and characterization of host factors contributing to the course of HIV infection, including immunological defense mechanisms and genetic factors, will be crucial for our understanding of the immunopathogenesis of HIV infection and for the development of immunotherapeutic and prophylactic strategies.

The structure of HIV-1

HIV-1 is a retrovirus and belongs to the family of lentiviruses. Infections with lentiviruses typically show a chronic course of disease, a long period of clinical latency, persistent viral replication and involvement of the central nervous system. Visna in sheep, simian immunodeficiency virus (SIV) in monkeys, or feline immunodeficiency virus (FIV) in cats are typical examples of lentivirus infections.

Using electron microscopy, HIV-1 and HIV-2 resemble each other strikingly. However, they differ with regard to the molecular weight of their proteins, as well as having differences in their accessory genes. HIV-2 is genetically more closely related to SIV found in sootey mangabeys (SIVsm) rather than HIV-1 and it is likely that it was introduced into the human population via monkeys. Both HIV-1 and HIV-2 replicate in CD4 T cells and are regarded as pathogenic in infected persons, although the immune deficiency may be less severe in HIV-2-infected individuals.

The morphologic structure of HIV-1

HIV-1 viral particles have a diameter of 100 nm and are surrounded by a lipoprotein membrane. Each viral particle contains 72 glycoprotein complexes, which are integrated into this lipid membrane, and are each composed of trimers of an external glycoprotein gp120 and a transmembrane spanning protein gp41. The bonding between gp120 and gp41 is only loose and therefore gp120 may be shed spontaneously within the local environment. Glycoprotein gp120 can be detected in the serum as well as within the lymphatic tissue of HIV-infected patients. During the process of budding, the virus may also incorporate different host proteins from the membrane of the host cell into its lipoprotein layer, such as HLA class I and II proteins, or adhesion proteins such as ICAM-1 that may facilitate adhesion to other target cells. The matrix protein p17 is anchored to the inside of the viral lipoprotein membrane. The p24 core antigen contains two copies of HIV-1 RNA. The HIV-1 RNA is part of a protein-nucleic acid complex, which is composed of the nucleoprotein p7 and the reverse transcriptase p66 (RT). The viral particle contains all the enzymatic equipment that is necessary for replication: a reverse transcriptase (RT), an integrase p32 and a protease p11 (Gelderbloom 1993) (Fig. 1).

The organization of the viral genome

Most replication competent retroviruses depend on three genes: gag, pol and env: gag means “group-antigen”, pol represents “polymerase” and env is for “envelope” (Wong-Staal 1991) (Fig. 2). The classical structural scheme of a retroviral genome is: 5’LTR-gag-pol-env-LTR 3’. The LTR (long terminal repeat) regions represent the two end parts of the viral genome, that are connected to the cellular DNA of the host cell after integration and do not encode for viral proteins. The gag and env genes code for the nucleocapsid and the glycoproteins of the viral membrane; the pol gene codes for the reverse transcriptase and other enzymes. In addition, HIV-1 contains six genes (vif, vpu, vpr, tat, rev and nef) in its 9kB RNA that contribute to its genetic complexity. Nef, vif, vpr and vpu were classified as accessory genes in the past, as they are not absolutely required for replication in vitro. However, the regulation and function of these accessory genes and their proteins have been studied and characterized in more detail over the past few years. The accessory genes nef, tat and rev are all produced early in the viral replication cycle.

Tat and rev are regulatory proteins that accumulate within the nucleus and bind to defined regions of the viral RNA: TAR (transactivation-response elements) found in the LTR; and RRE (rev response elements) found in the env gene, respectively. The tat protein is a potent transcriptional activator of the LTR promoter region and is essential for viral replication in almost all in vitro culture systems. Cyclin T1 is a necessary cellular cofactor for tat (Wei 1998). Tat and rev stimulate the transcription of proviral HIV-1 DNA into RNA, promote RNA elongation, enhance the transportation of HIV RNA from the nucleus to the cytoplasm and are essential for translation. Rev is also a nuclear export factor that is important for switching from the early expression of regulatory proteins to the structural proteins synthesized later on.

Figure 1: Structure of an HIV virion particle. For detailed explanations see text.

 

Figure 2: HIV and its genes. For detailed explanations see text.

 

Nef has been shown to have a number of functions. It may induce downregulation of CD4 and HLA class I molecules (Collins 1998) from the surface of HIV-1-infected cells, which may represent an important escape mechanism for the virus to evade an attack mediated by cytotoxic CD8 T cells and to avoid recognition by CD4 T cells. Nef may also interfere with T cell activation by binding to various proteins that are involved in intracellular signal transduction pathways (Overview  in: Peter 1998). In SIV-infected rhesus macaques, an intact nef gene was essential for a high rate of virus production and the progression of disease. HIV-1, with deletions in nef, was identified in a cohort of Australian long-term non-progressors (Kirchhoff 1995). However, more recent reports indicate that some of these patients are now developing signs of disease progression including a decline of CD4 T cells. Thus, although deletions of the nef gene may slow viral replication, they cannot always prevent the eventual development of AIDS.

Vpr seems to be essential for viral replication in non-dividing cells such as macrophages. Vpr may stimulate the HIV LTR in addition to a variety of cellular and viral promoters. More recently, vpr has been shown to be important for the transport of the viral pre-integration complex to the nucleus (Overview in: Miller 1997) and may arrest cells in the G2 phase of the cell cycle.

Vpu is important for the virus “budding” process, because mutations in vpu are associated with persistence of viral particles at the host cell surface. Membrane molecules such as tetherin (CD317) can bind vpu-deficient HIV-1 and prevent viral release. Thus, vpu can be considered as a viral escape mechanism in order to antagonise this effect (Neil 2009) and appears to be of great importance for the evolution of the pandemic virus (Sauter 2009). Vpu is also involved when CD4-gp160 complexes are degraded within the endoplasmic reticulum and therefore allows recycling of gp160 for the formation of new virions (Cullen 1998).

Some recent publications have highlighted a new and important role for vif in supporting viral replication (Mariani 2003). Vif-deficient HIV-1 isolates do not replicate in CD4 T cells, some T cell lines (non-permissive cells) or in macrophages. Vif-deficient isolates are able to enter a target cell and initiate reverse transcription, but synthesis of proviral DNA remains incomplete. In vitro fusion of permissive and non-permissive cells leads to a non-permissive phenotype, suggesting that the replication of HIV depends on the presence or absence of a cellular inhibitor. This endogenous inhibitory factor was identified as APOBEC3G (Sheehey 2002). APOBEC3G (apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3G) belongs to a family of intracellular enzymes that specifically deaminate cytosine to uracil in mRNA or DNA resulting in an accumulation of G-to-A mutations that lead to degradation of viral DNA. By forming a complex with APOBEC3G, vif blocks the inhibitory activity of APOBEC3G (Fig. 3a).

Of interest, the antiviral activity of APOBEC3G is highly conserved among various species, whereas the blockade of APOBEC3G by vif is highly specific for HIV. HIV-1 vif does not complex to murine or rhesus APOBEC3G. In the absence of vif, APOBEC3G is incorporated into newly formed viral particles and in subsequently infected target cells synthesis of proviral DNA is blocked (Fig 3b). In contrast, in the presence of vif, APOBEC3G is complexed, degraded and not incorporated in newly formed virions. APOBEC3G is expressed in lymphocytes and macrophages representing the primary target cells of HIV infection. In DC, the activation status of the cells influences the amount of APOBEC3G. Upon DC maturation there is an increase of APOBEC3G expression (Pion 2006).

Figure 3a: HIV wild-type infection: vif interacts with APOBEC3G, binds to APOBEC3G and prevents its incorporation in newly formed viruses.
Figure 3b: Vif-deleted HIV isolates fail to inhibit intracellular APOBEC3G, which is then incorporated into new viruses and interferes with reverse transcription in the target cell.

There are still a lot of open questions regarding the regulation of intracellular APOBEC3G. For example, whether there is a critical amount of intracellular APOBEC3G that restricts HIV infection in the presence of vif, or whether genetic polymorphisms of APOBEC3G exist that may potentially affect the course of disease, is not clear. In addition, the enzymatic function of intracellular APOBEC3G in lymphocytes may depend on the cellular activation status (Chiu 2005). Meanwhile, the epitopes by which vif and APOBEC3G interact with each other have been characterized and the pathway of intracellular degradation of the APOBEC3G-vif complex explored. Of note, specific inhibitors that block the interaction of vif and APOBEC3G or that interfere with the intracellular degradation of APOBEC3G could represent promising future treatments. In principle, blockade of cellular structures will likely be associated with a minimal risk that the development of resistance might compromise the efficacy of an antiviral agent. Therefore, targeting vif and APOBEC3G probably represents an interesting therapeutic track.

In summary, these data explain not only why vif is essential for HIV replication but also show why HIV replication is species-specific. Another cellular factor (see below) has also been discovered which explains species specificity of HIV replication.

The crucial role for APOBEC3G or other cytidine deaminases might not be restricted to HIV-1. An accumulation of G-to-A mutations was also demonstrated in various HBV isolates. In vitro, the accumulation of HBV DNA is dramatically reduced in the presence of APOBEC3G but co-transfection with vif can revert this inhibition.

The HIV replication cycle

HIV entry

CD4 as a primary receptor for HIV

CD4 is a 58 kDa monomeric glycoprotein that can be detected on the cell surface of about 60% of T lymphocytes, on T cell precursors within the bone marrow and thymus, and on monocytes and macrophages, eosinophils, dendritic cells and microglial cells of the central nervous system. The extracellular domain of the CD4 on T cells is composed of 370 amino acids; the hydrophobic transmembrane domain and the cytoplasmic part of CD4 on T cells consist of 25 and 38 amino acids, respectively. Within the extracellular part of CD4, four regions D1-D4 have been characterized that represent immunoglobulin-like domains. Residues within the V2 region of CD4 (amino acids 40-55) are important for the bonding of gp120 to CD4 and this region overlaps the part of the CD4 where its natural ligands, HLA class II molecules, bind.

The identification of the gp120 binding site on the CD4 receptor of CD4 T cells stimulated attempts to use soluble CD4 (sCD4) to neutralize the circulating virus in patients, the aim being the inhibition of viral spread. However, it became evident that even though laboratory viral isolates were easily neutralized by sCD4, neutralization of primary patient-derived isolates was not achieved.

In contrast, sCD4 was able to induce conformational changes within the viral envelope that promoted the infection of target cells.

CD4 attaches to the T cell receptor complex (TCR) on CD4 T cells and binds to HLA class II molecules on antigen-presenting cells. The binding of gp120 to CD4 is not only a crucial step for viral entry, but also interferes with intracellular signal transduction pathways and promotes apoptosis in CD4 T cells (Banda 1992). In the past couple of years, the idea of blocking CD4 as the primary cellular receptor of HIV has regained an interest. PRO542 represents a genetically engineered tetravalent CD4-IgG2 fusion protein that not only inhibits viral replication in vitro, but also shows an impressive antiviral efficacy in patients with high viral load that were in the initial clinical trials (see the chapter on ART).

CD4, as a primary and necessary receptor for HIV-1, HIV-2 and SIV, was already characterized in 1984 (Dalgleish 1984). However, experiments using non-human cell lines transfected with human CD4, showed that expression of human CD4 on the cell surface of a non-human cell line was not sufficient to allow entry of HIV. Therefore the existence of additional human co-receptors necessary for viral entry was postulated. On the other hand, some laboratory HIV-1 isolates, as well as some HIV-2 and SIV isolates are able to infect human cells independently from CD4. Interestingly, monoclonal antibodies against CD4-induced conformational (CD4i) epitopes to bind to the gp120 of CD4-independent viruses. This observation suggests that the gp120 of CD4-independent viruses already exposes the regions that are necessary for co-receptor recognition and binding and therefore binding to CD4 is not a prerequisite of entry for these viruses. CD4-independent viruses are easy to neutralize using the serum of HIV-infected patients, suggesting that the immune response selects against CD4-independent viruses (Edwards 2001).

Chemokine receptors as co-receptors for HIV entry

A milestone for the characterization of the early events leading to HIV-1 entry was an observation by Cocchi and co-workers in 1995. CD8 T cells from HIV-infected patients are able to suppress viral replication in co-cultures with HIV-infected autologous or allogenic CD4 T cells, and this is independent from their cytotoxic activity (Levy 1996). Cocchi identified the chemokines MIP-1a, MIP-1ß and Rantes in supernatants from CD8 T cells derived from HIV-infected patients, and was able to show that these chemokines were able to suppress replication in a dose-dependent manner of some, but not all, viral isolates tested (Cocchi 1995). MIP-1a, MIP-1ß and Rantes are ligands for the chemokine receptor CCR5, and a few months later several groups were able to show that CCR5 is a necessary co-receptor for monocytotropic (M-tropic) HIV-1 isolates (Deng 1996, Doranz 1996, Dragic 1998). A few weeks earlier, the chemokine receptor CXCR4 (fusin) was described as being the co-receptor used by T cell-tropic (T-tropic) HIV isolates (Feng 1996). Monocytotropic (M-tropic) HIV-1 isolates are classically those viruses that are most easily propagated in macrophage cultures, are unable to infect T cell lines (i.e., immortalized T cells), but are able to easily infect primary T cells from peripheral blood samples. Conversely, T cell-tropic HIV-1 isolates have classically been identified as being those that are easily propagated in T cell lines, and grow poorly in macrophages, but are also able to easily infect primary T cells from peripheral blood samples. It should be noted that both M-tropic and T-tropic HIV-1 variants can easily infect primary human non-immortalized T cells in vitro.

Chemokines (Chemotactic cytokines) and their receptors have been previously characterized with regard to their role in promoting the migration (chemotaxis) of leukocytes and their pro-inflammatory activity. They are proteins of 68-120 amino acids which depend on the structure of their common cysteine motif, and may be subdivided into C-X-C (a-chemokines), C-C (ß-chemokines) and C-chemokines. Chemokines typically show a high degree of structural homology to each other and may share the receptors they bind to. Chemokine receptors belong to the group of receptors with seven transmembranic regions (7-transmembrane receptors) and are intracellularly linked to G-proteins.

SDF-1 (stromal cell-derived factor 1) was identified as the natural ligand of CXCR4 and is able to inhibit the entry of T-tropic HIV-1 isolates into activated CD4 T cells. Rantes (regulated upon activation T cell expressed and secreted), MIP-1a (macrophage inhibitory protein) and MIP-1ß represent the natural ligands of CCR5 and are able to inhibit the entry of M-tropic HIV-1 isolates into T cells. A schematic model is depicted in Fig. 4. T-tropic HIV-1 isolates mainly infect activated peripheral blood CD4 T cells and cell lines and use CXCR4 for entry into the CD4-positive target cell. M-tropic isolates are able to infect CD4 T cells, monocytes and macrophages, and depend on the use of CCR5 and CD4 for viral entry.

The interaction of gp120 and the cellular receptors is now understood in more detail. Gp120 primarily binds to certain epitopes of CD4. Binding to CD4 induces conformational changes in the gp120 that promote a more efficient interaction of the V3 loop of gp120 with its respective co-receptor. Membrane fusion is dependent on gp120 co-receptor binding. Gp41, as the transmembrane part of the envelope glycoprotein gp160, is crucial for the fusion of the viral and host cell membrane. Similar to influenza hemagglutinin, it was postulated that consequent to the binding of gp120 to CD4, a conformational change is induced in gp41 that allows gp41 to insert its hydrophobic NH2 terminal into the target cell membrane. Gp41 has been compared to a mouse trap and a crystallographic analysis of the ectodomain of gp41 seems to confirm that (Chan 1997). The identification of crucial amino acid sequences for this process was used to synthesize peptides that bind to gp41 within the domains, are critical for the induction of conformational changes, and that may inhibit membrane fusion.

T-20 is the first of several peptides that bind to gp41 and has been tested in clinical trials for suppressing viral replication (see chapter on ART). T-20 is available as a therapeutic option for patients with advanced HIV. One disadvantage of T-20 is that it must be taken subcutaneously twice a day.

Using transfected cell lines, besides CCR5 and CXCR4, other chemokine receptors, such as CCR3, CCR2, CCR8, CCR9, STRL33 (“Bonzo”), Gpr 15 (“Bob”), Gpr 1, APJ and ChemR23, were identified and shown to be used for entry by certain HIV isolates (Deng 1997, Liao 1997). HIV-1 may also bind to certain integrins such as α4β7 and perturb cell function and migration (Arthos 2008). APJ may represent a relevant co-receptor within the central nervous system. Despite this broad spectrum of potentially available co-receptors, CCR5 and CXCR4 seem to represent the most relevant co-receptors for HIV-1 in vivo.

Figure 4: Inhibition of viral entry of CCR5-utilizing (monocytotropic) and CXCR4-utilizing (T cell tropic) HIV isolates by the natural ligands of the chemokine co-receptors CCR5 and CXCR4.

The importance of CCR5 as the predominant co-receptor for M-tropic HIV isolates is underscored by another observation. The majority of individuals with a genetic defect of CCR5 are resistant to infection with HIV-1 (Liu 1996). In vitro experiments show that lymphocytes derived from these individuals are resistant to HIV-1 infection using M-tropic isolates but not to infection with T-tropic isolates. Lymphocytes from these individuals do not express CCR5 on their cell surface and genetically have a 32-basepair deletion of the CCR5 gene. Worldwide, a few patients have been identified that have acquired HIV-1 infection despite a homozygous deletion of the CCR5. As expected, all of them were infected with CXCR4-using HIV-1 isolates. In epidemiological studies, the allelic frequency of the CCR5 gene deletion is 10-20% among Caucasians, particularly amongst those of Northern European descent. The frequency of a homozygous individual is about 1% in Caucasians (Dean 1996). Studies conducted on African or Asian populations, however, do not find this 32-basepair deletion, suggesting that this mutation arose after the separation of these populations in evolutionary history.

Individuals that are heterozygous for the 32-bp deletion of the CCR5 show a decreased expression of CCR5 on the cell surface and are more frequently encountered within cohorts of long-term non-progressors compared to patients who have a rapid progression of disease (Dean 1996). In addition, HIV-infected individuals who are heterozygous for the 32-bp deletion, have a slower progression to AIDS, a better treatment response to ART and lymphoma incidence is decreased. These data demonstrate that the density of CCR5 on the cell surface is not only a limiting factor for replication of HIV in vitro but in vivo as well.

In addition to the 32-bp deletion, other genetic polymorphisms with regard to chemokine receptors (CCR2) or their promoters (CCR5) have been described. Based on the occurrence of these polymorphisms within defined patient cohorts, they are associated with a more rapid or a more favorable course of disease, depending on the particular polymorphism (Anzala 1998, Winkler 1998). More recent studies shed light on the impact CCL3L1-CCR5 genotypes have not only on disease progression but also on response to antiretroviral therapy (Ahuja 2008). The mechanism appears to be independent of viral entry mechanisms but rather related to the quality of the cell-mediated immune response (Dolan 2007).

In patients who have a rapid progression of disease (rapid drop in CD4 T cell count), virus isolates that use CXCR4 as a predominant co-receptor tend to be frequently isolated from their cells in comparison to patients with a stable CD4 T cell count. The expression of co-receptors on CD4 lymphocytes depends on their activation level.

CXCR4 is mainly expressed on naive T cells, whereas CCR5 is present on activated and effector/memory T cells. During the early course of HIV-1 infection, predominantly M-tropic HIV-1 isolates are detected. Interestingly, M-tropic HIV-1 isolates are preferentially transmitted regardless of whether or not the source predominantly harbors T-tropic isolates. At present, it remains unclear whether this in vivo preference of M-tropic HIV-1 isolates is determined by selected transportation of M-tropic isolates by sub-mucosally located dendritic cells or whether the local cytokine/chemokine milieu favors the replication of M-tropic viruses. Intriguing studies suggest that M-tropic HIV-1 viruses are able to ‘hide’ more easily from the immune system by replicating in macrophages, in comparison to T-tropic viruses, thus giving them a survival advantage in the infected individual.

The blockade of CCR5, therefore, seems to represent a promising target for therapeutic intervention (Fig. 5). In vitro, monoclonal antibodies to CCR5 (2D7 and others) are able to block the entry of CCR5-using HIV isolates into CD4 T cells and macrophages. Small molecule inhibitors of CCR5 have been designed and preliminary clinical trials (see chapter on ART) demonstrate a significant reduction of plasma viremia in HIV-infected patients (Fätkenheuer 2005). In vitro studies as well as experiments using SCID mice do suggest that blockade of CCR5-using isolates may alter their tropism towards increased usage of CXCR4 (De Clercq 2001).

Small molecule inhibitors such as T-22, ALX40-4C or AMD3100 are able to inhibit CXCR4 and are also subject to preclinical and clinical trials (see chapter on ART). Other CCR5 inhibitors have been used as mucosal microbicides in monkey models and could represent a future preventive approach (Veazey 2005).

Strategies are currently being developed to modulate expression of chemokine receptors. Intrakines are chemokines that stay within the cytoplasm and are able to capture and bind to their corresponding receptor on its way to the cell surface (Chen 1997). Short interfering RNA (siRNA) represents a new molecular tool that is able to selectively inactivate target genes. Double-stranded RNA is split by the enzyme dicer-1 into short pieces (21-23-mers). These oligomers may complementarily bind to longer RNA sequences that are subsequently degraded. This strategy is currently employed in plants and used for its antiviral activity. The use of siRNA against CCR5 can prevent the expression of CCR5 in vitro and targeting of gag can effectively block viral replication (Song 2005).

Figure 5: Strategies to block infection by CCR5-tropic HIV. Blockage of CCR5 on the cell surface by non-agonistic ligands (A) or monoclonal antibodies (B). Alternatively, CCR5 cell surface expression can be reduced by siRNA or intrakine. For further details see text.

Although the therapeutic use of chemokine receptor blockers seems promising, a lot of questions still remain unanswered. In knockout mice it was demonstrated that the absence of CXCR4 or SDF-1 is associated with severe defects in hematopoiesis and in cerebellar development (Zou 1997). Currently, it remains unclear whether the blockade of CXCR4 in postnatal or adult individuals may affect other organ systems.

Post-fusion events

Following membrane fusion the viral core uncoats into the cytoplasm of the target cell. Alternatively, receptor-mediated endocytosis and dynamin-dependent fusion with intracellular compartments (Miyauchi 2009) can lead to viral inoculation. HIV can enter into rhesus lymphocytes but replication is stopped before or during early reverse transcription. This intracellular blockade is mediated by a cellular factor, TRIM5α, a component of cytoplasmic bodies whose primary function is not yet understood. TRIM5α from various species exhibits differential inhibition on various retroviruses. For example, TRIM5α from rhesus macaques, TRIM5αrh, more profoundly inhibits HIV replication than human TRIM5α, whereas SIV (simian immunodeficiency virus) which naturally infects Old World monkeys, is less susceptible to either form of TRIM5α, thus explaining in part the species specificity of HIV for human cells (Stremlau 2004). TRIM5α from human cells or non-human primates is able to inhibit replication of other lentiviruses and represents a novel cellular resistance factor whose definitive biological significance has yet to be fully characterized. TRIM5α serves as a mechanism for intracellular recognition and activation of the unspecific immune response (Pertel 2011), but it is unclear how exactly TRIM5α blocks reverse transcription and it has been hypothesized that TRIM5α interferes with the incoming virus capsid protein, targeting it for ubiquitination and proteolytic degradation.

HIV-1 entry into quiescent T cells is comparable to HIV-1 entry into activated T cells, but synthesis of HIV-1 DNA remains incomplete in quiescent cells (Zack 1990). The conversion of viral RNA into proviral DNA, mediated by the viral enzyme reverse transcriptase (RT), occurs in the cytoplasm of the target cell and is a crucial step within the viral replication cycle (Fig. 6). Blockade of the RT by the nucleoside RT inhibitor AZT was the first attempt to inhibit viral replication in HIV-1 infected patients. Today, numerous nucleoside, nucleotide and non-nucleoside RT inhibitors are available for clinical use and have broadened the therapeutic arsenal substantially since the mid-eighties.

Life cycle of HIV

Reverse transcription occurs in multiple steps. After binding of the tRNA primers, synthesis of proviral DNA occurs as a minus-strand polymerization starting at the PBS (primer binding site) and extending to the 5’ repeat region as a short R/U5 DNA. The next step includes degradation of RNA above the PBS by the viral enzyme RNAase H and a template switch of the R/U5 DNA with hybridization of the R sequence at the 3’ RNA end. Now the full length polymerization of proviral DNA with degradation of the tRNA is completed. Reverse transcription results in double-stranded HIV DNA with LTR regions (long terminal repeats) at each end.

HIV-1 enters into quiescent T cells and reverse transcription may result in the accumulation of proviral, non-integrating HIV DNA. However, cellular activation is necessary for integration of the proviral HIV DNA into the host cell genome after transportation of the pre-integration complex into the nucleus. Cellular activation may occur in vitro after stimulation with antigens or mitogens. In vivo, activation of the immune system is observed after antigen contact or vaccination or during an opportunistic infection. In addition, evidence is emerging that HIV-1 gp120 itself may activate the infecting cell to enhance integration. Besides monocytes, macrophages and microglial cells, latently infected quiescent CD4 T cells that contain non-integrated proviral HIV DNA represent important long-lived cellular reservoirs of HIV (Chun 1997), and cellular microRNAs contribute to HIV-1 latency in resting primary CD4 T lymphocytes (Huang 2007). Since natural HIV-1 infection is characterized by continuing cycles of viral replication in activated CD4 T cells, viral latency in these resting CD4 T cells likely represents an accidental phenomenon and is not likely to be important in the pathogenesis of HIV. This small reservoir of latent provirus in quiescent CD4 T cells gains importance, however, in individuals who are treated with ART, since the antivirals are unable to affect non-replicating proviruses – the virus will persist in those cells and be replication-competent to start new rounds of infection if the drugs are stopped. It is the existence of this latent reservoir that has prevented ART from entirely eradicating the virus from infected individuals (Chun 2005).

Until recently it was not clear why HIV replicates poorly in quiescent CD4 T cells. The cellular protein Murr1 that plays a role in copper metabolism is able to inhibit HIV replication in unstimulated CD4 T cells. Murr1 was detected in primary resting CD4 T cells and interferes with activation of the transcription factor NFkB by inhibiting the degradation of IkBa. IkBa prevents NFkB from migrating to the nucleus, especially after cytokine stimulation (e.g., TNFa). Because the HIV LTR region has multiple sites for NFkB, preventing NFkB migration to the nucleus should inhibit HIV replication. Inhibition of Murr-1 by siRNA is associated with HIV replication in quiescent CD4 T cells (Ganesh 2003). Persistence of HIV in quiescent CD4 T cells and other cellular reservoirs seems one of the main reasons why eradication of HIV is not feasible and why current therapies fail to achieve viral eradication (Dinoso 2009, Lewin 2011). If it is ever possible to achieve, a more detailed knowledge of how and when cellular reservoirs of HIV are established and how they may be targeted is of crucial importance for the development of strategies aiming at HIV eradication.

Cellular transcription factors such as NF-kB may also bind to the LTR regions. After stimulation with mitogens or cytokines NF-kB is translocated into the nucleus where it binds to the HIV LTR region, thereby initiating transcription of HIV genes. Transcription initially results in the early synthesis of regulatory HIV-1 proteins such as tat or rev. Tat binds to the TAR site (transactivation response element) at the beginning of the HIV-1 RNA in the nucleus and stimulates transcription and the formation of longer RNA transcripts. Rev activates the expression of structural and enzymatic genes and inhibits the production of regulatory proteins, therefore promoting the formation of mature viral particles. The proteins coded for by pol and gag form the nucleus of the maturing HIV particle, while the gene products coded for by env form the gp120 spikes of the viral envelope. The gp120 spikes are synthesized as large gp160 precursor molecules and are cleaved by the HIV-1 protease into gp120 and gp41. The gag proteins are also derived from a large 53 kD precursor molecule, from which the HIV protease cleaves the p24, p17, p9 and p7 gag proteins. Cleavage of the precursor molecules by the HIV-1 protease is necessary for the generation of infectious viral particles, and therefore the viral protease represents another interesting target for therapeutic blockade. The formation of new viral particles is a stepwise process: a new virus core is formed by HIV-1 RNA, gag proteins and various pol enzymes and moves towards the cell surface. The large precursor molecules are cleaved by the HIV-1 protease, which results in the infectious viral particles budding through the host cell membrane. During the budding process, the virus lipid membranes may incorporate various host cell proteins and become enriched with certain phospholipids and cholesterol. In contrast to T cells, where budding occurs at the cell surface and virions are released into the extracellular space, the budding process in monocytes and macrophages results in the accumulation of virions within cellular vacuoles.

The replication of retroviruses is prone to error and is characterized by a high spontaneous mutation rate. On average, reverse transcription results in 1-10 errors per genome and per round of replication. Mutations can lead to the formation of replication-incompetent viral species. Mutations that cause drug resistance may also accumulate, which, provided that there is selective pressure due to specific antiretroviral drugs and incomplete suppression of viral replication, may become dominant.

In addition, viral replication is dynamic and turns over quickly at an average rate of 109 new virus particles produced and subsequently cleared per day. Thus, within any individual, because of the extensive viral replication and mutation rates, there exists an accumulation of many closely-related virus variants within the population of viruses, referred to as a viral quasispecies. Selection pressure happens not only due to certain drugs, but also due to components of the immune system such as neutralizing antibodies or cytotoxic T cells (CTL).

HIV and the immune system

The role of antigen-presenting cells

Dendritic cells as prototypes of antigen-presenting cells

Dendritic cells, macrophages and B cells represent the main antigen-presenting cells of the immune system. Dendritic cells (DC) are the most potent inducers of specific immune responses and are considered essential for the initiation of primary antigen-specific immune reactions. DC precursors migrate from the bone marrow towards the primary lymphatic organs and into the submucosal tissue of the gut, the genitourinary system and the respiratory tract. They are able to pick up and process soluble antigens and migrate to the secondary lymphatic organs, where they activate antigen-specific T cells. Because DC have a crucial role in adaptive immunity, there is an increasing interest in using dendritic cells to induce or expand HIV-specific T cells. DC from HIV-infected patients have been purified, incubated with inactivated non-infectious HIV particles and subsequently used for vaccination (Lu 2004).

DC represent a heterogeneous family of cells with different functional capacities and expression of phenotypic markers depending on the local microenvironment and the stage of maturation. Immature DC have the capacity to pick up and process foreign antigens, but do not have great T cell stimulatory capacities. Mature DC show a predominant immunostimulatory ability. DC in tissues and Langerhans cells, which are specialized DC in the skin and mucosal areas, represent a more immature phenotype and may take up antigen. Once these DC have taken up the antigen, they migrate to the lymphoid tissues where they develop a mature phenotype.

The stimulation of CD8 T lymphocytes and the formation of antigen-specific cytotoxic T cells (CTL) depend on the presentation of a peptide together with MHC class I antigens. DC may become infected with viruses, for instance influenza. Viral proteins are then produced within the cytoplasm of the cell, similar to cellular proteins, then degraded to viral peptides and translocated from the cytosol into the endoplasmic reticulum, where they are bound to MHC class I antigens. These peptide-MHC class I complexes migrate to the DC surface. Alternatively, DC take up antigens for infected cells for presentation via MHC class I (Maranon 2004). Interestingly, the efficacy of this presentation of viral antigens is comparable whether or not DC themselves are productively infected. DC are rather resistant against productive HIV infection and intracellular recognition by DC contributes to innate immune repones following inoculation (Manel 2010).

The number of specific antigen-MHC class I complexes is usually limited and must eventually be recognized by rare T cell clones to a ratio of 1:100,000 or less. The T cell receptor (TCR) may display only a low binding affinity (1 mM or less). The high density of co-stimulatory molecules on the DC surface, however, enhances the TCR-MHC: peptide interaction allows efficient signaling to occur through the T cell and results in proliferation (clonal expansion) of the T cell. Virus-infected cells or tumor cells often do not express co-stimulatory molecules, and thus may not be able to induce a clonal expansion of effector cells. This underscores the importance of having a highly specialized system of antigen-presenting cells, i.e., DC, to prime T cells to expand and proliferate rapidly.

The interaction of dendritic cells and B/T cells

B and T lymphocytes may be regarded as the principle effector cells of antigen-specific immune responses. However, their function is under the control of dendritic cells. DC are able to pick up antigens in the periphery. These antigens are processed and expressed on the cell surface together with co-stimulatory molecules that initiate T cell activation. B cells may recognize antigen after binding to the B cell receptor. Recognition of antigen by T cells requires previous processing and presentation of antigenic peptides by DC and the intracellular half life time of peptides impacts on the CD8 T cell response (Lazaro 2011). T cells express different T cell receptors (TCR) that may bind to the peptide: MHC class I on the surface of dendritic cells to allow activation of CD8 T cells, or to the peptide: MHC class II molecules, to activate CD4 T cells. The ability of DC to activate T cells also depends on the secretion of stimulatory cytokines such as IL-12, which is a key cytokine for the generation and activation of TH1 and natural killer (NK) cells.

Only a few DC and small amounts of antigen are sufficient to induce a potent antigen-specific T cell response, thus demonstrating the immunostimulatory potency of DC. The expression of adhesion molecules and lectins, such as DC-SIGN, support the aggregation of DC and T cells, promote the engagement of the T cell receptor (TCR) and mutual infection and therby distribution of the virus within the host (Gringhuis 2010). DC-SIGN is a type C lectin that has also been shown to bind to lentiviruses such as SIV and HIV-1 and -2 by interaction of gp120 with carbohydrates (Geijtenbeek 2000). Mycobacteria and Dengue virus may also bind to DC-SIGN. In vivo, immunohistochemical studies show expression of DC-SIGN on submucosal and intradermal DC, suggesting an implication of DC-SIGN in vertical and mucosal transmission of HIV. The expression of DC-SIGN was shown to enhance the transmission of HIV to T cells and allows utilization of co-receptors if their expression is limited. Thus DC-SIGN may be a mechanism whereby HIV-1 is taken up by DC in the mucosal tissues. It is then transported by the DC to the lymphoid tissues where HIV-1 can then infect all the residing CD4 T cells (Lore 2005).

Lymphatic tissue as the site of viral replication

Viral replication within the lymphatic tissue is extensive in the early stages of the disease even in the hematopoietic system (Carter 2010, Embretson 2003, Pantaleo 1993).

During the initial phase of HIV-1 infection, there is a burst of virus into the plasma, followed by a relative decline in viremia. During this time, a strong HIV-1 specific cytotoxic T cell response is generated, which coincides with the early suppression of plasma viremia  in most patients. Virions are trapped by the follicular dendritic cell (FDC) network within the lymphoid tissue. Macrophages and activated and quiescent CD4 T cells are the main targets of infection. Permanent viral reservoirs, mainly in macrophages and latently infected CD4 T cells are established in the early phase of infection and probably represent the major obstacle so far to successful  eradication of HIV. During the whole course of infection with HIV-1, the lymphoid tissue represents the principle site of HIV-1 replication. The frequency of cells containing proviral DNA is 5-10 times higher in lymphoid tissue than in circulating peripheral mononuclear cells in the blood, and the difference in viral replication in lymphoid tissue exceeds that in the peripheral blood by about 10-100 times.

After entry of HIV-1 into a quiescent CD4 T cell and after completion of reverse transcription, the viral genome is represented by proviral unintegrated HIV DNA. In vitro experiments have shown that HIV-1 preferentially integrates into active genes (hot spots) (Schroder 2002). The activation of CD4 T cells is necessary for the integration of the HIV DNA into the host cell genome and is therefore a prerequisite for the synthesis of new virions. In this regard, the micromilieu of the lymphoid tissue represents the optimal environment for viral replication. The close cell-cell contact between CD4 T cells and antigen-presenting cells, the presence of infectious virions on the surface of the FDC, and an abundant production of pro-inflammatory cytokines such as IL-1, IL-6 or TNFα (Stacey 2009), promotes the induction of viral replication in infected cells and augments viral replication in cells already producing the virus. During later stages of the disease, micriobial translocation from the gut (Brenchley 2006, Klatt 2010, Estes 2010) contribute to systemic immune actiation, which in turn lead to loss of CD4 T cells (Ciccone 2010) and disease progress. It should be noted that both IL-1 and TNFα induce NF-kb, which binds to the HIV-1 LTR to promote proviral transcription. The importance of an antigen-induced activation of CD4 T cells is underlined by several in vivo and in vitro studies that demonstrate an increase in HIV-1 replication in association with tetanus or influenza vaccination or an infection with Mycobacterium tuberculosis (O’Brian 1995). Even though the clinical benefit of vaccination against common pathogens (e.g., influenza and tetanus) in HIV-1-infected patients outweighs the potential risk of a temporary increase in viral load, these studies indicate that in every situation where the immune system is activated, enhanced viral replication can also occur.

Patients on ART demonstrate a dramatic decrease in the number of productively infected CD4 T cells within the lymphoid tissue (Tenner-Racz 1998). However, in all patients so far, there persists a pool of latently infected quiescent T cells despite successful suppression of plasma viremia. It is these latently infected cells that may give rise to further rounds of viral replication if the antiviral drugs are stopped.

During the natural course of HIV-1 disease, the number of CD4 T cells slowly decreases while plasma viremia rises in most patients. If sequential analysis of the lymphoid tissue is performed, progression of the disease is reflected by destruction of the lymphoid tissue architecture and a decreased viral trapping. Various immunohistological studies indicate that the paracortex of the lymph nodes represents the primary site where HIV replication is initiated (Embretson 1993, Pantaleo 1993). Infection of the surrounding CD4 T cells, as well as the initiation of T cell activation by DC, contributes to the spreading of HIV-1 within the lymphoid environment. Similar to SIV infection in rhesus macaques, HIV infection at all stages of disease is associated with preferential replication and CD4 T cell destruction in the gut lamina propria and submucosa rather than in lymph nodes (Veazey 1998, Brenchley 2004). This is likely because the gut is predominantly populated by CCR5-expressing effector memory CD4 T cells, which are ideal targets for HIV replication compared to the mixed populations of CD4 T cells found in the lymph nodes. In addition, HIV-1 nef appears to have lost a function which can be found in SIV, where it leads to a down-regulation of CD3 and the T cell receptor, reducing chronic cell activation which would otherwise render them susceptible for apotosis (Schindler 2006). Several studies have demonstrated that, during acute infection, depletion of CD4+CCR5+ memory cells within the mucosa-associated lymphatic tissue is a hallmark of both HIV and SIV infection. Genome analysis of viral RNA in patients with acute HIV infection revealed that in about 80% of infected patients, viremia results from infection of a single virus particle (Keele 2008). In the early phase of SIV infection, up to 60% of all CD4 T cells within the intestinal lamina propria were shown to express viral RNA. Most of these cells are destroyed by direct and indirect mechanisms within a few days. Further disease progression seems to depend largely on the capacity of the host to reconstitute the pool of memory cells within the mucosa-associated lymphoid tissue. In view of this data, some researchers argue that initiation of ART during acute HIV infection is crucial in order to limit long-term damage to the immune system. However, in patients with exhaustion of lymphpoiesis despite effective long-term HIV therapy disease progression might occur (Sauce 2011).

Recent studies have also examined the effect of HIV infection on the thymus and its role in CD4 T cell depletion and homeostasis. Recent work has suggested that thymic output of CD4 T cells is decreased during HIV infection, particularly with older patients, and that this defect is due to abnormalities of intra-thymic proliferation of T cells, whose mechanism is still undefined, as thymocytes do not express CCR5 and should not necessarily be targets of HIV (Douek 2001).

The HLA system and the immune response to HIV

CD8 T cells recognize specific antigens (peptides) in context with HLA class I molecules on antigen-presenting cells, whereas CD4 T cells require the presentation of antigenic peptides in context with HLA class II molecules. The generation of an HIV-specific immune response is therefore dependent on the individual HLA pattern.

Antigen-presenting cells may bind HIV peptides in different ways on the HLA class I molecules. Therefore, CD8 T cells can be activated in an optimal or suboptimal way or may not be activated at all. Using large cohorts of HIV-1 infected patients, in whom the natural course of disease (fast versus slow progression) is known, HLA patterns were identified that were associated with a slow versus fast disease progression (Pereyra 2010). These studies suggest that the HLA type may be responsible for a benign course of disease in about 40% of patients with a long-term non-progressive course of disease. Homozygosity for HLA Bw4 is regarded as being protective. Patients who display heterozygosity at the HLA class I loci characteristically show a slower progression of immunodeficiency than patients with homozygosity at these loci (Carrington 1999). An initial study by Kaslow in 1996 demonstrated that HLA B14, B27, B51, B57 and C8 are associated with a slow disease progression, while the presence of HLA A23, B37 and B49 were associated with a rapid development of immunodeficiency (Kaslow 1996). All patients with HLA B35 had developed symptoms of AIDS after 8 years of infection. More recent studies suggest that discordant couples with a “mismatch” regarding the HLA class I have a protective effect towards heterosexual transmission (Lockett 2001).

In vitro studies in HLA B57-positive patients demonstrate that these patients display HLA B57-restricted CTL directed against HIV-1 peptides. There is evidence that the better immune responses in these patients is already determined during thymic selection (Kosmrlj 2010). However, it is possible that the identification of protective HLA alleles or HLA-restricted peptides in HIV-1-infected patients with a benign course of disease does not necessarily indicate that the same alleles or peptides are crucial for the design of a protective vaccine. CD8 T cells from HIV-1-exposed but uninfected African women recognize different epitopes than CD8 T cells from HIV-1-infected African women (Kaul 2001). This suggests that the epitopes that the immune system is directed against during a natural infection might be different from those that are protective against infection. In addition, the individual HLA pattern may affect the adaptive immune response and the evolution of viral escape mutations (Friedrich 2004, Leslie 2004). CTL from patients with HLA B57 and B58 may force the virus to develop certain mutations in gag that enable the virus to escape the CTL response, but these mutations result in a reduced replicative competence and thus lower viral load (Goepfert 2008). If such a virus is transmitted to another individual with a different HLA background, the virus may back-mutate to the original genotype and regain its full replicative competence because of the absence of CTL-mediated immune pressure.

HLA class II antigens are crucial for the development of an HIV-1-specific CD4 T cell response. Rosenberg (1997) was the first to show that HIV-1-infected patients with a long-term non-progressive course of disease had HIV-1-specific CD4 T cells that could proliferate against HIV-1 antigens. The identification of protective or unfavorable HLA class II alleles is less well elaborated than the knowledge about protective HLA class I alleles. Cohorts of vertically infected children and HIV-infected adults demonstrate a protective effect of HLA DR13 (Keet 1999).

KIR receptors (Killer cell immunoglobulin like receptors) represent ligands that bind to HLA class I antigens and by functioning as either activating or inhibiting receptors they regulate the activation status of NK cells. Polymorphisms of KIR genes were shown to correlate with slow or rapid progression of HIV disease, especially when the analysis includes known HLA class I polymorphisms (Fauci 2005). During HIV infection NK cells may not only be decreased but may also show a diminished cytolytic activity. Preliminary results suggest that low numbers of NK cells are associated with a more rapid disease progression .

In summary, various genetic polymorphisms have been identified that have an impact on the course of HIV disease. However, there is currently no rationale to recommend routine testing of individual patients or to base therapeutic decisions on genetic testing.

The HIV-specific cellular immune response

Cytotoxic T cells (CTL) are able to recognize and eliminate virus-infected cells. A number of studies clearly demonstrate that CTL are crucial for the control of HIV replication and have a substantial impact on disease progression once infection is established. However, there is little evidence to assume that CTL play a major role in primary protection.

In comparison to HIV-1-infected patients with a rapid decline in CD4 T cell numbers, patients with a long-term non-progressive course of disease (LTNP = long-term non-progressors) have high quantities of HIV-1-specific CTL precursors with a broad specificity towards various HIV-1 proteins. The different capacities of certain HLA alleles to present viral particles more or less efficiently and to induce a generally potent immune response may explain why certain HLA alleles are associated with a more rapid or a slower progressive course of disease (see above).

Individuals have been described, who developed CTL escape mutants after years of stable disease and the presence of a strong CTL response. The evolution of CTL escape mutants was associated with a rapid decline in CD4 T cells in these patients, indicating the protective role of CTL (Goulder 1997).

HIV-specific CTL responses have been detected in individuals exposed to, but not infected by HIV-1. Nef-specific CTL have been identified in HIV-1-negative heterosexual partners of HIV-infected patients and env-specific CTL have been found in seronegative healthcare workers after exposure to HIV-1 by needle stick injuries (Pinto 1995). Unfortunately, patients with a broad and strong CTL response do not seem to be protected from superinfection by a different but closely related HIV isolate (Altfeld 2002).

The presence of a CTL response is not correlated just with the suppression of plasma viremia during the initial phase of HIV infection. Patients who underwent structured therapy interruptions, especially when ART was initiated early following infection, demonstrated the appearance of HIV-specific CTL during the pauses. Goonetilleke (2009) demonstrated that the initial HIV-1 specific CTL response contributes significantly to the control of viremia during acute infection. Novel imaging techniques and animal models enabled for the first time the visualization of the interplay of immune cells with infected target cells and the contribution of this response for the infection control (Li 2009).

However, it is still unclear in most patients who exhibit a potent temporary CTL response why this CTL response diminishes later on (Pantaleo 2004). The appearance of viral escape mutants might explain why previously recognized epitopes are no longer immunodominant.

The nef protein may downregulate HLA class I antigens and therefore counteract the recognition of infected cells by CTL. In addition, the majority of infected individuals show detectable CTL responses. It is unclear why they are unable to control the virus. Interestingly, CTL from HIV-infected patients shows a lack of perforin and an immature phenotype in comparison to anti-CMV-directed effector cells (Harari 2002) even though the ability to secrete chemokines and cytokines is not impaired (Appay 2000). Another study provided evidence that the killing capacity of HIV-specific CTL was associated with the ability to simultaneously produce interferon-g and TNFa (Lichtenfeld 2004). Surface molecules such as PD-1 on CTL, which are transiently upregulated upon cell activation, may persist due to prolonged antigen presence. Persistent PD-1 expression, however, can result in CTL and helper cells (Said 2010) dysfunction and this effect can be restored by blockade of PD-1 and PD-1L interaction on DC by administration of anti-PD-1 antibodies. This restores CTL functions such as cytokine production and killing capacity (Trautmann 2006, Velu 2009).

CD8 T cells may also become infected with HIV (Bevan 2004) although this has not been demonstrated for HIV-specific CD8 T cells. It is unclear whether CD8 T cells temporarily express CD4 and which chemokine co-receptors mediate infection of these CD8 T cells.

Proliferation and activation of CTL is dependent on antigen-specific T cell help. Initiation of ART during primary HIV infection was associated with persistence of an HIV-specific CD4 T cell response that was not detected in patients analyzed during the chronic stage of disease (Rosenberg 1997). HIV-specific CD4 T cells are mainly directed against gag and nef-derived epitopes (Kaufmann 2004). HIV preferentially infects pre-activated CD4 T cells and as HIV-specific CD4 T cells are among the first cells to be activated during HIV infection, their preferential infection has been demonstrated (Douek 2002). Therefore, it is currently unclear whether the loss of HIV-specific CTL activity during the course of disease reflects an instrinsic defect of CTL or develops secondary to a loss of specific CD4 T cell help.

Various therapeutic vaccine strategies have been developed during the last few years and mostly tested in SIV-infected rhesus macaques aiming at inducing an SIV-specific CTL response that may alter the natural course of disease (McElrath 2010). Recently, a promising approach was published on a vaccine trial using autologous dendritic cells in SIV-infected rhesus macaques that were pulsed with inactivated SIV (Lu 2003). In contrast to the unvaccinated control group, monkeys that were vaccinated showed a dramatic decrease in viral load, and the development of anti-SIV-directed humoral and cellular immune responses. Meanwhile, a pilot trial has been initiated in a cohort of 18 HIV-infected antiretroviral-naive patients with stable viral load. The patients were vaccinated with autologous monocyte-derived dendritic cells that were pulsed with inactivated autologous virus. During the following 112 days, a median decrease of 80% of the viral load was observed and maintained for more than one year in 8 patients. In parallel, gag-specific CD8 T cells and HIV-specific CD4 T cells producing IFNg and/or interleukin-2 were detected (Lu 2004). Therapeutic vaccination using autologous dendritic cells appears to be a potential immunotherapeutic, but more controlled clinical studies are definitely needed.

In addition to the cytotoxic activities directed against HIV-infected cells, CD8 T cells from HIV-1 infected patients exhibit a remarkable, soluble HIV-1 inhibitory activity that inhibits HIV-1 replication in autologous and allogeneic cell cultures (Walker 1986). Despite multiple efforts, the identity of this inhibitory activity (CAF) has not been clarified, although chemokines such as MIP-1α, MIP-1ß or Rantes (Cocchi 1995), IL-16 (Baier 1995), the chemokine MDC (Pal 1997) and defensins may account for at least some of the inhibition.

The TH1/TH2 immune response

Depending on the secretion pattern of cytokines, CD4 T cells may be differentiated into TH1 and TH2 cells. TH1 CD4 T cells primarily produce interleukin-2 (IL-2) and IFNg, which represent the cytokines that support the effector functions of the immune system (CTL, NK-cells, macrophages). TH2 cells predominantly produce IL-4, IL-10, IL-5 and IL-6, which represent the cytokines that favor the development of a humoral immune response. Since TH1 cytokines are critical for the generation of CTLs, an HIV-1-specific TH1 response is regarded as being a protective immune response. Studies on HIV-exposed but non-infected individuals have shown that following in vitro stimulation with HIV-1 env antigens (gp120/gp160) and peptides, T cells from these individuals secrete IL-2 in contrast to non-exposed controls (Clerici 1991). Similar studies were undertaken in healthcare workers after needlestick injuries and in newborns from HIV-infected mothers. Although these observations may indicate that a TH1-type immune response is potentially protective, it should be considered that similar immune responses might have been generated after contact with non-infectious viral particles and therefore do not necessarily imply a means of protection against a replication-competent virus.

HIV-1 specific humoral immune responses

The association between an HIV-1-specific humoral immune response and the course of disease is less well characterized.

In an SIV model injection of an antibody cocktail consisting of various neutralizing antibodies is able to prevent SIV infection after a mucosal virus challenge (Ferrantelli 2004) indicating that primary protection is mainly dependent on a broad humoral immune response. Additional animal experiments suggest antibody concentrations in mucosal sites required to confer protection (Hessell 2009). This data suggests that HIV-specific antibodies are necessary for a preventive vaccine strategy. In contrast, B cell depletion by a monoclonal antibody directed against B cells in monkeys with already established SIV infection does not affect the course of plasma viremia (Schmitz 2003).

A slow progression of immunodeficiency was observed in patients with high titers of anti-p24 antibodies (Hogervorst 1995), persistence of neutralizing antibodies against primary and autologous viruses (Montefiori 1996) and lack of antibodies against certain gp120 epitopes (Wong 1993).

Long-term non-progressors with HIV tend to have a broad neutralizing activity towards a range of primary isolates and show persistence of neutralizing antibodies against autologous virus. At present, it is unclear whether the presence of neutralizing antibodies in LTNP represents part of the protection or whether it merely reflects the integrity of a relatively intact immune system. Individuals that have a substantial risk for HIV-1 infection, but are considered exposed yet non-infected by definition represent individuals with a lack of a detectable antibody response to HIV-1. This definition implies that a systemic humoral immune response may not represent a crucial protective mechanism. It has been shown that these individuals may demonstrate a local (mucosal) IgA response against HIV-1 proteins that are not detected by the usual antibody testing methods (Saha 2001). Thus, local IgA, rather than systemic IgG, may be associated with protection against HIV-1 infection. There is also some evidence that some anti-HIV-1 antibodies can enhance infection of CD4 T cells.

A number of older as well as recent studies have shown that neutralizing antibodies do exist in HIV-1-infected individuals, although there is a time lag in their appearance. That is, individuals will develop neutralizing antibodies to their own viruses over time, although by the time these antibodies develop the new viruses circulating in the individual’s plasma will become resistant to neutralization, even though the older ones are now sensitive to the current antibodies in the patient’s serum. Thus, the antibody response appears to be constantly going after a moving target, allowing viruses to escape continuously. Further understanding the mechanisms of humoral escape will likely lead to potential new therapies.

A few years ago, selected patients with advanced HIV infection were treated with plasma from HIV-infected patients at an earlier stage of the disease. No significant effect on the course of disease was notable (Jacobson 1998). The therapeutic application of neutralizing antibodies with defined specificity looked more promising, since a few acute and chronically infected patients were able to control their viral load at least temporarily after stopping antiretroviral therapy (Trkola 2005). Functionally, Fc receptors but not complement binding are important in antibody protection against HIV (Hessell 2007) and some neutralising antibodies recognise the CD4-binding site of gp120 (Li 2007). Neutralizing antibodies frequently recognize conserved epitopes with relevance for viral fitness (Pietzsch 2010) and research during the last years has revealed some of the specific structures and surface glycoproteins, which are targeted by neutralizing antibodies (Wu 2010, Zhou 2010). This more detailed understanding is very likely to advance vaccine strategies aiming to treat or to protect against HIV-infection.

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