Chapter 18: Retroviruses: Human T-Lymphotropic Virus, Human Immunodeficiency Virus, and Acquired Immunodeficiency Syndrome

Retroviruses are enveloped, single-stranded, positive-sense RNA viruses. These viruses are known as retroviruses because they encode an enzyme called reverse transcriptase, which converts the RNA genome into a double-stranded DNA copy that subsequently becomes integrated into the host chromosome. The discovery of reverse transcriptase in 1970 by two American virologists, David Baltimore and Howard Temin, earned them a Nobel Prize in Medicine. There are two major groups of retroviruses that infect humans: the oncoretroviruses (onco-, “related to a tumor”) and the lentiviruses (lenti-, “slow”). There are several other groups of retroviruses that infect animals. Endogenous retrovirus sequences are found throughout the human genome. Like most enveloped viruses, all retroviruses are highly susceptible to factors that affect surface tension and are thus not transmissible through air, dust, or fomites under normal conditions, but instead require intimate contact with the infecting sources, such as bodily fluids, blood, and blood-derived products.

Members of the oncoretrovirus, a subgroup of retroviruses, have long been associated with a variety of cancers in animals, including leukemias, lymphomas, and sarcomas. However, an oncoretrovirus was discovered in the late 1970s that infects humans known as human T-cell lymphotropic virus type I (HTLV-I). It causes adult T-cell leukemia and lymphoma (ATLL), a rare malignancy found only in Japan, Africa, and the Caribbean, although serologic evidence shows that it also occurs in the United States and has raised the possibility of an association with some chronic neurologic conditions. A relative of HTLV-I, HTLV-II has been associated with a few rare cases of T-cell malignancies, including hairy cell leukemia, but its precise role in these diseases remains unclear.

The most important disease resulting from a human retrovirus infection is called acquired immunodeficiency syndrome (AIDS), which is caused by a lentivirus known as human immunodeficiency virus (HIV). There are two types: HIV-1 and HIV-2, which cause AIDS. A devastating disease worldwide, for which there is no permanent cure or preventive vaccine for protection, AIDS has spurred unprecedented research efforts to determine the nature and immunopathogenic mechanisms of the virus in the hope of finding more and new effective drugs and a preventive AIDS vaccine. Most of our present knowledge of HIV is derived from studies on HIV-1, which is the major cause of AIDS worldwide. In 2008, two French virologists, Françoise Barré-Sinoussi and Luc Montagnier, shared the Nobel Prize in Medicine for their work on the discovery of HIV-1, the virus that causes AIDS.

Oncoretroviruses are not cytolytic in the sense that they do not kill the cells that they infect, but rather they transform the cells by different mechanisms (see Chapter 7 and HTLV section of this chapter) and continue to produce low levels of new virus indefinitely. With lentivirus infections, the host cell–virus relationship is different. Lentiviruses can apparently persist in infected hosts for long periods of time in a clinically latent state. Over time, the virus becomes highly cytopathic and kills CD4+ T-cells (lymphocytes), causing impairment of the host immune defenses followed by development of AIDS and opportunistic infections. The prototype of this type of lentivirus is HIV-1. The second type of HIV, HIV-2, also causes immunodeficiency in humans that develops slowly and tends to be milder and mainly found in West Africa. HIV-1 is the causative agent of AIDS and found in most of the HIV infected people worldwide.

HIV-1 can remain clinically latent in most infected patients without causing viral latency in untreated patients, which means that virus is produced at low levels without serious disease, but when allowed to replicate in the absence of effective immune response and other factors, high levels of virus are produced causing CD4+ T-lymphocyte cell death and AIDS. Although HIV-1 can infect a variety of human cell types, such as T-lymphocytes, monocytes/macrophages, dendritic cells, Langerhans cells, and microglia/glial cells, its most drastic effects appear to result from destruction of the CD4+ T-lymphocytes, which play a central role in the capacity of the host to mount effective and protective immunologic responses to a wide range of infections.

VIROLOGY

STRUCTURE

All retroviruses are remarkably similar in their basic virion composition and structure. The virion structure of HIV-1 is depicted in Figure 18–1. The virion size is about 100 nm in diameter, and because it contains two copies of the RNA genome, it is diploid. The RNA genome is coated with the NC protein, and the RNA–protein complexes are enclosed in a capsid (CA, also called p24) composed of multiple subunits in an icosahedral symmetry, which is covered by a membrane-associated matrix (MA, also called p17) protein. Like all enveloped viruses, the lipid bilayer membrane is acquired during budding from the host cell plasma membrane, but the surface (SU, also called gp120) and transmembrane (TM, also called gp41) glycoproteins found in the envelope are virally encoded. Gp120 binds to CD4 receptor and coreceptor and CCR5 or CXCR4 (chemokine receptor) binds on CD4+ T-cells and other cells. In addition to the structural proteins shown in Figure 18–1, the virion core contains three virus-specific proteins (enzymes) that are essential for viral replication: Reverse transcriptase (RT), protease (PR), and integrase (IN). The relation between the viral genes found in all retroviruses (gag, pol, and env) and the proteins they encode are presented in Table 18–1. Some retroviruses, including HTLV and HIV-1, encode additional regulatory and accessory proteins. Based on SU gp120 variable region/loop 3 (V3 loop) sequence, HIV-1 that binds to CD4 and CXCR4 is called X4 (T-lymphotropic) HIV-1, whereas HIV-1 that binds to CD4 and CCR5 is called R5 (Macrophage tropic) HIV-1. Some HIV-1 isolates are also X4/R5 HIV-1 (dual tropic).

RETROVIRAL REPLICATION CYCLE

Figure 18–2 depicts the life cycle of a typical retrovirus (eg, HIV-1) and serves to illustrate the many unique aspects of retroviral replication that are targets for current antiviral agents and could be potential targets of new and effective therapeutic intervention.

Viral Entry

Retroviral virions are adsorbed to cellular membrane receptors through an interaction of viral surface protein and cellular receptors and enter the cell by direct fusion of the viral envelope with the plasma membrane of the host cell. For HIV-1, the virion attachment protein is the SU glycoprotein, gp120, and the cellular receptor is the CD4 molecule with one of the chemokine receptors, CXCR4 or CCR5, acting as a coreceptor. These receptors and coreceptors are expressed primarily on the plasma membrane of CD4+ T-lymphocytes, but also on cells of the monocyte–macrophage lineage, and some other target cells such as Langerhans cells, dendritic cells, and certain brain cells. The naïve CD4+ T-lymphocytes express higher levels CXCR4 and somewhat lower levels of CCR5. However, the mucosal memory CD4+ T-lymphocytes, monocytes/macrophages, Langerhans cells and others express higher levels of CCR5 but lower levels of CXC4. Inhibitors of CCR5 coreceptor are available to be used in combination HIV-1 therapy. Early in infection, the HIV-1 isolates in patients are R5 because R5 viruses that use CCR5 coreceptor are predominantly transmitted to recipients. The emergence of syncytia-forming HIV-1 variants that use the CXCR4 coreceptor are X4 viruses that appear to correlate with rapid advancement to AIDS. The HIV-1 transmembrane TM protein gp41 is responsible for fusion of the viral and cell membranes, leading to entry of the virion core complex into the cytoplasm of the cell. Fusion inhibitor to gp41 function is a peptide-based antiviral agent approved as a part of combination therapy when other first-line drugs have failed.

HIV-1 can also infect cells that lack the CD4 surface molecule such as certain brain cells and other cells types with a low efficiency, apparently because the chemokine receptors in combination with the fusion-inducing activity of the TM protein is sufficient in these cases to promote entry. Fusion activity may also play an important role in amplification of the effects of the virus infection, particularly during the later stages of the infection, because infected cells expressing viral glycoproteins in their membranes readily fuse with uninfected CD4+ T-lymphocytes to form large syncytia. This process appears to provide a means for cell-to-cell transmission of the virus that bypasses the usual extracellular phase and may contribute to the overall depletion of CD4+ T-lymphocytes in an infected person.

Viral Postentry Events

Among the RNA viruses, retroviral replication is unique because it involves reverse transcription. Soon after the entry of the viral core into the cytoplasm of the infected cell, there is partial uncoating and the viral RNA is reverse transcribed (converted) into a complementary DNA (cDNA) by the action of reverse transcriptase enzyme, the virion-associated RNA-dependent DNA polymerase. The cDNA is converted into double-stranded DNA by the action of the DNA-dependent DNA polymerase activity of the same reverse transcriptase enzyme. The viral RNA template is removed from the RNA–DNA hybrid by RNAase H activity of the same reverse transcriptase enzyme. The overall process is referred to as reverse transcription. Currently, there are several antiviral agents that are inhibitors of reverse transcriptase enzyme (nucleoside and nonnucleoside reverse transcriptase inhibitors) used in combination therapy (as the first line of drugs) to treat HIV-1 infection. Following reverse transcription, the resultant linear DNA molecule circularizes and makes a preintegration complex with the help of viral and host factors. The preintegration complex enters the nucleus and integrates more or less at random sites into the host cell chromosome catalyzed by viral integrase enzyme. The integration process is highly specific with respect to the viral DNA, and two base pairs are generally lost from each end of the DNA. The choice of a target site for integration into the cellular DNA appears, however, to be nearly random but preferably in actively transcribed host genes. Once the viral genetic information has been converted to DNA and integrated, it essentially becomes part of the cellular genome, and the cell is permanently infected. The viral genome, called the provirus, is therefore replicated and faithfully inherited as long as the infected cell continues to divide. Integrase inhibitors have been developed and approved as a part of combination HIV-1 therapy.

Special sequences contained within the RNA are duplicated during the reverse transcription process so that the integrated provirus contains identical long terminal repeats (LTRs) at its ends (Figure 18–3). The LTR sequences contain the appropriate promoter, enhancer, and other signals required for transcription of the viral genes by the host RNA polymerase II. Transcription produces a full-length RNA genome and one or more spliced mRNAs. For HIV-1, a series of spliced mRNAs are produced that encode envelope proteins and a series of viral regulatory and accessory proteins. Gag and Gag-Pol precursors are encoded by full-length RNA. Unlike most retroviruses, HIV-1 and the other lentiviruses apparently exert considerable control over whether the primary transcripts are allocated to full-length RNA or are spliced to produce mRNAs (see text that follows). With the exception of these regulatory and accessory proteins, all retroviral proteins are initially translated as polyproteins that are subsequently processed by proteolysis into the individual protein molecules. Although the HIV-1 envelope precursor proteins (gp160) are cleaved into gp120 and gp41 by host cell protease, the enzyme responsible for cleavages of Gag and Gag-Pol precursors into capsid proteins and enzymes, respectively, is the virus-specific protease (PR) that is encoded by the pol gene of HIV-1. HIV-1 protease inhibitors are approved for use as part of combination HIV-1 therapy.

Of all the known retroviruses, HIV-1 possesses the most error-prone reverse transcriptase. The consequence of this high error rate is that each time the viral RNA is reverse transcribed, three to four new mutations are introduced into the resulting DNA. Because the process of transcription of the integrated proviral DNA to produce new viral genomes is also error-prone, mutant genomes accumulate rapidly over the course of an infection. The end result is a quasispecies that accounts for the many nucleotide differences observed between different isolates (even from the same infected individual) and for the variability of the SU envelope protein gp120. It may explain, in part, the failure of the immune system to control the infection, the increases in viral virulence that appear to occur during the course of the infection, and the difficulty of developing an effective vaccine.

RETROVIRAL GENES

The genome organization of different types of retroviruses is shown in Figure 18–4 (see also Table 18–1). All retroviruses contain the same structural genes in the order of gag–pol–env genes. The gag (group-specific antigen) gene encodes the structural proteins (matrix-MA, capsid-CA, nucleocapsid-NC) of the virus and, in some animal retroviruses, the protease. The pol (polymerase) gene in human retroviruses and HIV-1 encodes the protease (PR), reverse transcriptase (RT), and the integrase (IN). The env (envelope) gene encodes the two membrane glycoproteins found in the viral envelope, SU gp120 and TM gp41. HIV-1 gp120 has five variable regions (V1-V5) and several constant regions (C1-C5). The CD4-binding domains on gp120 are localized in the constant regions, whereas the coreceptor (CXCR4/CCR5) binding regions on gp120 are confined in the variable region 3 (V3 loop). The V3 region is also the principal neutralizing domain of the virus, and therefore contributes to antigenic variation and varying degrees of neutralization. However, gp41 is embedded in the envelope and mediates fusion of the viral envelope with the plasma membrane at the time of viral infection and less variable than gp120.

A comparison of the genetic makeup of HIV-1 with that of a typical retrovirus (Figure 18–4) reveals a larger number of genes and a much more complex organization. HIV-1 contains, in addition to the gag, pol, and env genes, an array of other genes (tat, rev, nef, vif, vpr, and vpu). Expression of these genes requires mRNA splicing, and all apparently encode proteins that serve regulatory or accessory roles during the infection (see text that follows). HTLV-I encodes the regulatory proteins, Tax and Rex, which are analogous to the HIV-1 Tat and Rev proteins. The names of the genes that have been best characterized and the proteins and functions they determine are listed in Table 18–2.

VIRUS ASSEMBLY AND RELEASE

Once all the viral proteins are made, the process of virus assembly proceeds. The Env gp 120 and gp41 are expressed on cell surface. The nucleoprotein complexes of the Gag and Gag-Pol polyproteins with viral genomic RNA are created, where NC protein of Gag polyprotein binds to packaging site on the 5′ end of viral RNA. The MA protein of Gag polyprotein interacts with its C terminus to the NC-RNA-Gag-Gag-Pol complex and on the N terminus to the C terminus of g41 that is expressed in conjunction gp120 onto plasma membrane. This NC-RNA-Gag-Gag-Pol complex buds out of the plasma membrane with gp120 and gp41 on cell surface. The next step includes the morphogenesis involving proteolytic processing of Gag and Gag-Pol polyproteins by HIV-1 protease enzyme into various Gag proteins (MA, CA, NC) and Pol enzymes (PR, RT, IN), making it a complete infectious virus particle. If protease inhibitors are used as part of combination therapy, then Gag and Gag-Pol polyproteins will not be processed and mature infectious virus particles will not be made.

ROLES OF HIV-1 REGULATORY AND ACCESSORY PROTEINS

HIV-1 encodes a complex array of regulatory and accessory proteins that appear to be involved in viral replication, pathogenesis, and disease progression. Antivirals against these proteins may aid in improving HIV-1 treatment. These proteins also appear to interact with cellular factors to modulate the infection differently in different host cells. The roles of the two HIV-1 regulatory genes, tat and rev, and the four accessory genes, nef, vpu, vpr, and vif, are discussed later and summarized in Table 18–2.

The products of the tat and rev regulatory genes are the Tat and Rev proteins, respectively. Both proteins are essential for viral replication. When the infected T-lymphocyte is stimulated, for example, by antigen presentation, Tat and Rev play a positive role in promoting viral gene expression. Tat is a transcriptional activator that acts at a sequence near the beginning of the viral mRNA in the LTR, called Tat-acting responsive (TAR) element, to recruit cellular proteins to help the RNA polymerase to complete efficient transcription and make HIV-1 RNA of the HIV-1 proviral genome. In the absence of Tat, the host RNA polymerase initiates the transcription at the LTR promoter, but transcription is prematurely terminated leading to the production of a short, dead-end RNA.

The Rev protein is posttranscriptional transactivator that acts at the level of mRNA splicing and transport. Normally, unspliced RNAs are retained in the nucleus, and only multiply spliced mRNAs that encode Tat, Rev, and Nef are transported to the cytoplasm for translation. For the synthesis of proteins such as Env, Vif, Vpr, and Vpu that are made from singly spliced mRNAs, and the Gag and Pol polyproteins from the unspliced genomic RNA, it is necessary to transport these proteins mRNAs to the cytoplasm. Transport of these singly spliced mRNAs or unspliced RNAs is accomplished by Rev binding to an RNA sequence within the env gene called the Rev-responsive element (RRE). The Rev-RRE interaction exports the singly spliced mRNAs or unspliced RNAs from the nucleus to cytoplasm for translation. By promoting translation of the virion structural proteins and some of the accessory proteins, Rev turns up late gene expression that leads directly to a high rate of virus production.

The Nef accessory protein interferes with immune recognition of infected cells. Nef causes the internalization and degradation of the CD4 protein, which likely prevents superinfection and the formation of complexes between the cellular receptor and newly synthesized virions. Nef also downregulates the cell surface major histocompatibility complex (MHC) I molecules, which may prevent killing of infected cells by cytotoxic T-lymphocytes (CTLs). In addition, virions produced in the absence of the Nef protein are at least partially blocked at some step before integration. The combination of these and perhaps other effects allows the Nef protein to play an essential pathogenic role in an infected individual.

The Vpu protein of HIV-1 appears to play two separate roles during the late stages of infection. In the absence of Vpu, the Env protein forms complexes with CD4 in the endoplasmic reticulum and fails to reach the plasma membrane of the cell. One of the roles of Vpu is to target the destruction of CD4 in the endoplasmic reticulum to allow for incorporation of Env into newly synthesized virions. The second role of Vpu is to promote the release of virions from the infected cell. The most likely mechanism is that Vpu counteracts the function of a host factor, BST-2 (bone marrow stromal antigen 2, CD137 or tetherin). BST-2 tethers HIV-1 to the cell and prevents virus release, and thus has antiviral activity.

The Vpr protein is required for efficient viral replication in resting T-cells and monocytes/macrophages. Several possible roles for Vpr in HIV-1 replication have been suggested, including modest transactivation of HIV-1 LTR, enhancement of the nuclear migration of the preintegration complex in the newly infected nondividing cells, inhibition of establishment of chronic infection, arrest of cells in the G2/M phase of the cell cycle, and inducing latent cells into a high level of virus production. Furthermore, successful infection of nondividing cells such as macrophages and resting T-lymphocytes requires Vpr to allow the newly synthesized viral DNA to reach the nucleus and be integrated into the cellular DNA.

HIV-2 encodes Vpx instead of Vpu. Vpx has homology to Vpr and shares the functions of Vpr. The functions of Vpr and Vpx have been segregated, including Vpr maintaining the ability to induce G2 arrest, whereas Vpx retains the ability to enhance infection of nondividing cells such as macrophages.

Vif (virion infectivity factor) increases the infectivity of HIV-1 in primary T-cells and monocytes/macrophages in culture. In the absence of Vif, the virus fails to complete reverse transcription in these cell types. Vif also inhibits an RNA editing enzyme, APOBEC3G (apolipoprotein B, a member of innate immune system), which causes hypermutation in HIV-1 DNA after reverse transcription and inhibiting viral replication.

Superimposed on this complex regulatory network is the fact that the viral promoter contains elements that are sensitive to specific cellular transcription factors. This observation may help explain why virus production in CD4+ T-lymphocytes is greatly increased when the cells are activated. Clearly, the outcome of an HIV-1 infection is determined by a complex interplay among very large number of different factors.

HUMAN IMMUNODEFICINEY VIRUS AND ACQUIRED IMMUNODEFICIENCY SYNDROME

EPIDEMIOLOGY

AIDS was first recognized in the United States in 1981, when it became apparent that an unusual number of rare skin cancers (Kaposi sarcoma) and opportunistic infections were occurring among male homosexuals. These patients were found to have a marked reduction in CD4+ T-lymphocytes and were subject to a wide range of opportunistic infections normally controlled by an intact immune system. The disease was found to progress relentlessly to a fatal outcome and was first identified in male homosexuals, hemophiliacs, who were receiving blood-derived coagulation factors, and injection drug users.

Retrospective serologic studies with material saved from patients in various studies indicate that HIV-1 infection was already occurring in Africa in the 1950s and in the United States in the 1970s. In 1985, HIV-2 was found to be endemic in parts of West Africa and to cause a milder immunodeficiency at a slower pace. To date, this virus has been relatively restricted geographically, although HIV-2 infections have occurred in the Western Hemisphere. Therefore, HIV-1 will be referred as HIV in this section, as it is the major cause of AIDS worldwide.

Transmission

HIV is transmitted between humans in several ways: sexually, parenterally, vertically, and by exposure to contaminated bodily fluids, blood or blood-derived products. The virus has been demonstrated in bodily fluids particularly in high titers in semen, vaginal and cervical secretions, rectal fluids, and breast milk. HIV is mainly transmitted in the United States through anal or vaginal sex, the highest risk is through receptive anal sex (risk 1.38%), although insertive anal sex (risk 0.11%) also spread the virus. In vaginal sex, both partners have risk of transmission but the receptive partner (female), has a higher risk (0.08%) than insertive partner, male (risk 0.04%), although the risk is lower than receptive anal sex. Worldwide, penile-vaginal sex is the major route of transmission. People sharing needles or syringes for intravenous drug use can spread the virus at a higher risk of 0.63%, whereas percutaneous needle-stick risk is 0.23% and exposure of HIV-infected blood and fluids to mouth, eye, nose or nonintact skin risk is 0.1%. The risk of HIV transmission through contaminated blood transfusion is extremely high (risk 92.5%), but the blood supply in the United States and other developed countries is rigorously tested. HIV is less commonly transmitted through needle stick or sharp objects for health-care workers. In extremely rare cases, HIV has been shown to be transmitted by oral sex, receiving blood and blood products (prescreened for HIV) or organs, contact with broken skin, biting, deep mouth kissing with sores. HIV transmission can be reduced by condom usage, circumcision, and ART in infected people. Mother-to-child transmission can occur prepartum (via transplacental route), intrapartum (through birth canal), and postpartum (through breast milk). It is important to note that ART during pregnancy has significantly reduced the risk of mother-to-child transmission of HIV by less than 1%.

Infection is facilitated by breaks in epithelial surfaces, which provide direct access to the underlying tissues or bloodstream. The relative fragility of the rectal mucosa and the large numbers of sexual contacts are probable contributing factors to the predominance of the disease among promiscuous MSM. HIV is transmitted in penile to vaginal sex to females by vaginal or cervical routes, despite natural barriers, such as multicellular layers of squamous epithelial cells of vaginal mucosa and antimicrobial activity of cervicovaginal secretions. The risk of transmission further increases with the disruption of integrity of the vaginal or rectal mucosa because of dry or traumatic sex and other infectious and inflammatory diseases. Once the virus is deposited in the vaginal or rectal mucosa, the virus can also traverse the mucous layer and probably reach the dendritic projections of Langerhans cells followed by infection of submucosal cells such as macrophages, T-lymphocytes, and dendritic cells.

Transmission due to blood or blood-products transfusion and organ transplantation has been significantly reduced in the United States and other developed countries due to rigorous HIV testing and screening for infectious agents. However, transmission by blood is now largely associated with sharing of needles and syringes by injecting drug users, and this has been an increasing source of the disease. In some areas of the world, the seroprevalence of HIV positivity among injecting drug users has been as high as 70%. Transmission of infection to healthcare workers through accidental needle-sticks that are potentially contaminated is very low (~ 0.23%). Nevertheless, transmission has occurred from both clinical and laboratory exposure, and extreme care in handling needles, sharps, and so on, is necessary. Transmission does not occur through day-to-day nonsexual contact with infected individuals or through insect vectors, because of the fragility of the virus and the need for direct mucosal or blood contact. As described earlier, HIV can be found in most bodily fluids. While HIV is found in saliva, transmission has not been documented, whereas HIV found in breast milk is readily transmitted to infants in the absence of ART.

Occurrence

Globally by the end of 2015, 36.7 million (34.0-36.8 million) people, including 17.8 million women and 1.8 million children were living with HIV, 2.1 million people (150 000 children, 50% lower than 2010) were newly infected with HIV in 2015 (no declines among adults since 2010), and 1.1 million people died of AIDS in 2015 (45% decline since 2005). Since the start of the epidemic until the end of 2015, 78 million people have been infected and 35 million people have died. More importantly, 18.2 million people have access to ART by June 2016. In 2015, 77% of pregnant women with HIV had access to antiretroviral treatment. Although sub-Saharan Africa has 25.6 million or 70% of all HIV-infected people in the world, about 5.1 million people are living with HIV in South, Southeast and East Asia at the end of 2015. After sub-Saharan Africa and Asia and Pacific, the most heavily affected area regions where 1% of the people are living with HIV in 2015 are the Caribbean, Eastern Europe, and Central Asia. By the end of 2015, 230 000 people were living with HIV in the Middle East and North Africa. One of the striking trends of the HIV epidemic is that 45% of infected people are between the ages of 15 and 24 years.

In the United States, approximately 1.2 million people are living with HIV, including 44% blacks/African American, 32% whites, 21% Hispanics, and about 1.3% Asians/Pacific Islanders, and American Indians. Males accounted for 76% of the HIV-infected population, and more than one-half million people have died with HIV/AIDS. New HIV infections in the recent years in the United States have remained relatively stable at around 50 000 per year. The highest prevalence rates (63%) have been in men who have sex with men (MSM) followed by high-risk heterosexual contact (25%), intravenous drug users (8%), and those infected with both male-to-male and injection drug use (3%). The overall rate of HIV perinatal (mother-to-child) transmission with ART in the United States has been less than 1%.

In contrast to the situation in the United States and Western Europe, heterosexual transmission is the primary route of transmission in Africa and Asia, where there is an approximately equal distribution of infection and disease between the sexes. This may be due to a high incidence in these areas of ulcerative genital lesions caused by other sexually transmitted diseases. These lesions facilitate passage of virus into the tissues of others during intercourse. In central and eastern Europe, where there is an emerging epidemic, the most common risk factor is intravenous drug use.

AIDS has been reported in more than 186 countries. The rate of new infection has dropped by 35% between 2000 and 2015 and HIV-related deaths by 28%. The sharpest declines in new infection by 41% was observed in the African countries between 2000 to 2014. However, the epidemics in Americas have remained unchanged. In European region, the number of newly HIV-infected people has increased since 2000, especially in eastern Europe. The number of people living with HIV in Russia is estimated to be 1.5 million, about 1% of its population. Between 2000 to 2014, the number of newly HIV-1 infected people has almost doubled in the Eastern Mediterranean region. In the South-East Asia region where HIV infection was exploding until 2009, the number of new HIV-1 infection has decreased and stabilized. In China, there are 0.5 million people living with HIV in 2014 with 115 000 new infections and 21 000 deaths. There are 2.1 million people living with HIV in India, with 86 000 new infections and 68 000 deaths in 2015, including a 32% decline in new infection in recent years. The declining or stabilizing infections in various regions of the world is attributed to access to ART.

HIV-1 Clades and Geographic Distribution

Based on genetic variation, three classes of HIV-1 have developed worldwide, including M (major), O (outlying), and N (new). However, class M accounts for more than 90% of all HIV-1 cases globally and is further classified into several subtypes or clades, including A to H and recombinants. In addition, the demographic distribution of individuals infected with particular clades is becoming heterogeneous with the progressing pandemic. However, several clades predominate in a given region of the world, including clade B (Americas, Europe, and Australia), clade C (India and South Africa), clade E (Southeast Asia), and most major clades and recombinants (Africa). Among all clades circulating worldwide, clade C is found in more than 50% of HIV-1–infected people. The interclade variation in the envelope gene is in the range of 20% to 30%, whereas intraclade variation is 10% to 15%. There is also some argument that certain clades may have increased risk of transmission and progress to AIDS more rapidly than others. Understanding the immunopathogenesis of the emerging HIV-1 clades is key to vaccine development.

PATHOGENESIS

HIV infection is typically characterized by: (1) an inefficient transmission of HIV (common route: anal or vaginal sex); (2) an acute phase of intense viral replication and dissemination to lymphoid tissues (antiretroviral syndrome; flu- or mononucleosis-like illness in infected individuals); (3) activation of innate and adaptive immune response but unable to contain the highly replicating and mutating virus; (4) a chronic (persistent) asymptomatic phase (clinical latency) of continued viral replication and immune activation; and (5) an advanced phase of marked depletion of CD4 T-lymphocytes (immune deficiency) leading to development of AIDS (opportunistic infections). Figure 18–5 summarizes the immunopathogenic events of HIV infection. Although the pathogenesis of HIV infection is very complex, the following factors are likely to be important in the disease-causing process.

Infection

Sexual transmission of HIV following exposure of infectious virus in semen or mucosal surfaces represents the common route of HIV transmission worldwide (other routes of HIV transmission are discussed earlier. The initial target of HIV is the CD4 molecule and a chemokine receptor (CCR5), particularly on the surface of monocytes/macrophages, Langerhans cells, and mucosal CD4+ helper T-lymphocytes, as a minor genotype of HIV (single founder virus) with R5 phenotype is predominantly transmitted from person to person. The first cell type to be infected is most likely the Langerhans cell or macrophages via CD4 and CCR5. The virus replicates in these cells, and these cells could serve as a reservoir for continued expansion of the infection to other cell types, especially CD4 or macrophages or T-lymphocytes (the major target cells) by cell-to-cell fusion. In addition, dendritic cells (DC-SIGN) also play an important role in transferring HIV to CD4 T-lymphocytes. HIV productively replicates in the genital mucosal CD4 T-lymphocytes (CD4+/CCR5+) and migrates via draining lymph nodes to gut-associated lymphoid tissue (GALT) and replicates and depletes memory CD4+ T-lymphocytes (CD4+/CCR5+) in intestinal lamina propria. HIV then disseminates to other secondary lymphoid tissue to establish stable viral reservoirs. At this time (2-4 weeks after transmission), a majority of patients experience flu- or mononucleosis-like illness (acute retroviral syndrome). During the early phase of infection, aggressive viral replication occurs in the absence of immune response and the concentration of HIV reaches 10 million copies per milliliter, which can be detected in blood as early as 7 to 28 days after transmission. There is a depletion of CD4+ T-lymphocytes in the peripheral blood and a massive depletion of CD4+ T-lymphocytes in the GALT. The immune system mounts a response that lags behind the high viral load and is unable to completely control viral replication. However, the viral load decreases as the virus establishes a set point in infected patients, which means the virus continues to replicate and mutate while avoiding the immune response. There is also a rebound of CD+ T-lymphocytes in the peripheral blood. This asymptomatic phase is also referred to as clinical latency.

The virus can also more efficiently infect T-lymphocytes, cells that express CD4 and CXCR4, which is seen in late stages of HIV disease. HIV can infect a wide range of CD4+ cells, including renal and gastrointestinal epithelium and brain astrocytes. The mechanism for infection of non–CD4-bearing cells is unknown, but may involve coreceptors such as CCR5 or CXCR4.

Infected monocytes may participate in breakdown of the blood–brain barrier, allowing monocytes to infiltrate the central nervous system (CNS). These infected monocytes differentiate into perivascular macrophages and become the resident cells harboring HIV in the CNS. Although CNS disturbance is a part of fully developed AIDS, it is not clear whether they are a direct result of infection of these cells or mediated by cytokines from infected macrophages and T-lymphocytes.

Following transmission, HIV replicates in CD4+/CCR5 cells and the predominant phenotype of HIV is R5 in infected people initially, whereas the highly replicating and mutating virus late in infection becomes X4, which replicates more efficiently in CD4 T-lymphocytes, causing cytopathic effects.

Kinetic studies of changes in viral load with antiviral therapy demonstrated that the half-life of HIV in plasma is 5 to 6 hours and an estimated 10 billion HIV particles are produced every day in an infected individual. In other words, more than 50% of the viral load measured on any given day has been produced in the last 24 hours. Because 99% of the viral load is produced by cells that were infected within the last 48 to 72 hours, cell turnover must be equally rapid. Indeed, when similar kinetic studies are performed on changes in CD4 cell counts, it is estimated that up to 1 billion CD4+ cells are produced per day in response to the infection and that the half-life of these cells is only 1.6 days.

Clinical Latency or Chronic Phase

Following infection and establishment of a viral set point, the long asymptomatic period (clinical latency or chronic phase) occurs despite active virus replication in the host. Several factors can terminate the long clinical latency period of HIV. Mutations occur during viral replication, which appear to enhance induction of virulent forms of the virus (conversion of R5 to X4), with increased cytopathic capacity and altered cell tropisms. Thus, the mutated forms of HIV isolated from later stages of disease (X4) infect a broader range of cell types and grow more rapidly than those isolated in the asymptomatic period (R5). Initially, it was believed that little or no viral replication occurred during this latent period, but studies of lymph nodes of individuals with early asymptomatic disease have shown intense immunologic reactions within the lymphoid tissue at early stages of disease. This implies that the immune system is capable of controlling the virus to some degree early in the course of disease, an ability that is later lost as the disease progresses over time. Figure 18–5 shows the temporal changes in viral load, anti-HIV immune responses, and total CD4+ T-cell counts during various stages of HIV infection.

Following clinical latency, various studies have shown that the level of free HIV in the plasma increases in direct relation to the stage of disease. Individuals with early-stage disease have less than 10 infectious virions per milliliter of plasma, whereas those in late-stage disease have between 100 and 1000/mL. These studies imply that either viral replication was increasing during later stages of disease as a result of more virulent mutations and/or the immune system had lost its ability to clear free virus as the disease progresses. However, current HIV treatment has changed these scenarios.

Immune Activation

HIV infection causes a generalized immune activation, including production of proinflammatory cytokines (TNF-α, interleukin-1 [IL-1], IL-6, IL-12) and chemokines, INF-α and lipopolysaccharides (LPS). One of these factors, LPS, is a potent activator of macrophages and dendritic cells to release proinflammatory cytokines during acute infection, most likely by translocation of microbial product (LPS) by disruption of intestinal barrier of GALT infection. The role of INF-α and TNF-α is described later.

Immune Response and its Failure to Eliminate HIV

Early control of HIV infection is achieved by innate immunity. Soon after infection, dendritic cells respond through recognition of viral products (viral RNA) by pattern recognition receptors (toll-like receptors 7 and 8) and releasing antiviral cytokines, INF-α and TNF-α, which inhibit viral replication and promote activation of immune response. Recent studies suggest that dendritic cells from females produce a higher level of INF-α than males probably resulting in a lower viral load set point in females compared with males. HIV Env gp120 binds to TLR9 causing activation of INF-α/β and NK cells that also provide early control of infection. Several other innate immune cells respond to HIV infection by releasing antiviral cytokines or factors through their distinct set of innate immune receptors. These cells include phagocytes (monocytes, macrophages, and dendritic cells that clear antigens), cytolytic cells (NK cells and neutrophils that destroy the pathogen or pathogen-infected cells) and professional antigen-presenting cells (APCs; dendritic cells that present antigens to adaptive immunity). Moreover, NK cells are activated by INF-α and IL-15 made by dendritic cells and kill HIV-infected cells to control early infection. However, HIV has found ways to interfere with the components of innate immunity and the infection proceeds.

The professional APCs, dendritic cells, make the transition from innate to adaptive immunity by presenting antigens to T-lymphocytes. HIV-specific CD8+ CTLs (cytotoxic T-lymphocytes) are generated that control plasma viremia by killing HIV-infected cells. The function of CTL is mediated by perforin that makes holes in the target cell through which granzyme can enter and destroy the infected cells. In addition, CD8+ T-lymphocytes express Fas ligand that can bind to Fas (CD95) on infected cells resulting in apoptosis-induced cell death. CD8+ T-lymphocytes produce INF-γ that creates an antiviral state and β-chemokines (MIP 1-α, MIP 1-β, and RANTES) that bind to CCR5 and reduce the ability of HIV to infect other uninfected cells. However, the emergence of CTL escape mutants, because of mutations generated due to continued viral replication, are unable to sustain suppression of viral replication. The B lymphocytes respond to HIV antigens by making neutralizing antibodies after the decline in the level of viremia. The B lymphocytes see antigens in the native form initially and later through interaction with HIV-specific CD4+ T-lymphocytes to generate neutralizing antibodies. These neutralizing antibodies neutralize cell-free virions. However, viral variants emerge that escape neutralization from antibody response allowing continued viral replication. HIV antibodies can be detected between 3-12 weeks after infection.

The CD4+ T-lymphocytes that make cytokines (especially IL-2) to help B lymphocytes and both CD4+ and CD8+ T-lymphocytes are impaired because CD4+ T-lymphocytes are infected and killed by HIV. In early infection, memory CD4+ T-lymphocytes are depleted; however, both memory and naïve CD4+ T-lymphocytes are depleted as the infection progresses.

Despite a robust immune response, the immune system fails to eliminate HIV from infected individuals. Several reasons could be attributed, including cell-to-cell spread of the virus that avoids recognition by the neutralizing antibodies; high mutation rates resulting in antigenic variation causing CTL and antibody escape variants; interference with cytokine production; suppression of MHC I and II; integration of proviral DNA into the host chromosome; establishment of persistent infection; and diminished ability of T-lymphocyte precursor to generate mature CD4+ and CD8+ T-lymphocytes. The immune system is unable to keep up with the pace of mutating virus, resulting in impaired T- and B-lymphocyte functions and immune deficiency.

Immune Deficiency

The primary immune deficiency in AIDS results from the reduction in the numbers and effectiveness of CD4+ helper T-lymphocytes, both in absolute numbers and relative to CD8+ T-lymphocytes. This is due to direct killing of CD4+ T-lymphocytes by the virus, but may also involve other mechanisms. These include secondary killing of uninfected (bystander) cells during cell fusion and syncytia formation, apoptosis, interference with T-cell maturation, autoimmune processes that lead to the elimination of CD4+ T-lymphocytes by opsonophagocytosis, and antibody-dependent cell-mediated cytotoxicity (ADCC) directed at gp120 expressed on the CD4+ cell surface. There are also functional defects in CD4+ T-lymphocytes affecting cytokine production and leading to inhibition of some macrophage functions.

Effects on CD4+ T-lymphocytes thus lead to a generalized failure of cell-mediated immune responses, but there is also an effect on antibody production due to polyclonal activation of B cells, possibly associated with other viral infections of these cells. This overwhelms the capacity of infected individuals to respond to specific antigens. The end result of these processes is a disturbance of immune balance that can give rise to malignancies as well as the susceptibility of AIDS patients to a range of opportunistic viral, fungal, and bacterial infections.

HIV Reservoirs

Following infection, HIV establishes persistent infection even in the presence of competent immune system. Whereas in the absence of ART (described later) infected individuals develop immune deficiency (described earlier) and opportunistic infections (described later), HIV persists in reservoirs (cells or tissues that harbor HIV) in the presence of effective ART. HIV reservoirs are the biggest hurdle in eradicating HIV from infected individuals by effective ART. There are two types of HIV reservoirs: lymphoid tissues (GALT and lymph nodes: many target cells for HIV and low penetration of ART) and cellular reservoirs (resting T-lymphocytes and monocytes/macrophages). In HIV-infected individuals undergoing successful viral suppression with ART, a small pool of resting CD4+ T-lymphocytes remain silently infected with HIV provirus that also provides a long-lived source of rebound viremia. The phenotype of these CD4+ T-lymphocytes includes central memory CD4+ T-lymphocytes (T_CM), transitional memory CD4+ T-lymphocytes (T_TM), and effector memory CD4+ T-lymphocytes (T_EM). Whereas T_CM that are long-lived quiescent T-lymphocytes present in lymph nodes might represent a latent reservoir for HIV-1, T_EM that are present in a high frequency in GALT may provide residual viral replication. Recent studies suggest that HIV can persist latently in CD34 stem cells in bone marrow, especially in those patients who do not start ART early after infection. Research continues to find ways to destroy HIV from these reservoirs.

CLINICAL ASPECTS

MANIFESTATIONS

In 1993, the CDC definition of AIDS stated that all patients who are HIV antibody positive and have CD4+ T-lymphocyte counts lower than 200/mm³ or less than 14% of total T-lymphocytes have the disease. HIV-1 infection is characterized as a three-stage process; (1) acute phase (flu- or mononucleosis-like illness, also known as acute retroviral syndrome), (2) clinical latency or chronic phase (asymptomatic with low level of HIV production), and (3) AIDS phase (immune deficiency, opportunistic infections).

• Stage 1: Acute Phase. After 2 to 4 weeks of infection, some infected individuals are asymptomatic, while other infected individuals develop a flu- or mononucleosis-like illness with many symptoms such as fever, chills, night sweats, sore throat, lymphadenopathy, arthralgias, fatigue, hepatosplenomegaly, and rash that lasts about 2 to 6 weeks. Sometimes a mild aseptic meningitis is also present. During this time, HIV RNA and antigen can be detected. Whether these early manifestations of infection occur or do not occur, the virus rapidly invades, persists, and integrates into the genome of some host cells, and the individual is thus infected for life.

• Stage 2: Clinical Latency or Chronic Phase. The initial infection is followed by an asymptomatic period (clinical latency, during which low level of virus is produced) that, in most cases, continues for years before the disease becomes clinically apparent. During this time, the virus can be isolated from blood, semen, and other bodily fluids and tissues. More than 60% of infected individuals remain in clinical latency for about 10 years after infection before they develop significant disease, and the number continues to increase thereafter if untreated. It is expected that nearly all HIV-infected persons eventually develop some clinical aspects of this infection if left untreated, although long-term (> 10 years) nonprogressors are well documented. Some infected individuals (5-10%) develop significant clinical diseases within few years after infection, if untreated, are referred as rapid progressors. Approximately 5% of infected, untreated patients show no decrease in CD4 counts over a period of more than 10 years, but ultimately many of these individuals begin to progress. Based on the availability of more specific and sensitive tests, HIV can be detected early in infection and potent ART can be initiated to suppress viral load, improve CD4 T-cell counts, and prevent infected patients developing clinical HIV disease, symptomatic AIDS.

• Stage 3: AIDS. As the disease progresses in untreated patients, the number of CD4+ T-lymphocytes declines. An increasing immunodeficiency, and opportunistic infections becoming more frequent, severe, and difficult to treat is considered AIDS. One of the best markers of the severity of AIDS is the absolute number of CD4+ T-lymphocytes. Those individuals with overt AIDS almost always have fewer than 200 CD4+ T-lymphocytes/mm³ of blood (normal = 500-1600/mm³), although opportunistic infections may occur with CD4+ T-cells greater than 200/mm³. Patients with AIDS at the late stage of HIV infection may experience many symptoms such as recurring fever, night sweats, rapid weight loss, diarrhea (for more than a week), sores in mouth or genitals, white patches on the tongue or oral mucous membranes (thrush), pneumonia, and some neurological disorders. Many infected people are tested for HIV-1 after experiencing these symptoms. If they are tested positive, viral load and CD4 T-cell counts, in addition to other blood work, are ordered, and treatment is initiated. Viral load and CD4 T-cells count are monitored to assess the progress of the treatment.

Patients with full-blown AIDS, who were untreated (no ART), experience a wide spectrum of infections depending on the severity of their immune deficiency and on the opportunistic organisms in their normal flora or those with which they come in contact (Table 18–3). Some clinical manifestations of AIDS may thus vary by locale. For example, disseminated histoplasmosis was a common complication in the Midwestern United States and disseminated coccidioidomycosis in the Southwestern United States, as was disseminated toxoplasmosis in France. These infections are uncommon in areas where the diseases are not endemic. The diversity and anatomic sites of infection vary among patients, and any one patient may have several infections. The most common infection is pneumocystosis, and approximately 50% of the AIDS patients who do not receive ART or prophylaxis for pneumocystosis develop Pneumocystis jirovecii pneumonia. In the past, about 25% of all patients with AIDS developed Kaposi sarcoma, but the number of cases has been falling in the United States despite increasing numbers of cases of AIDS. The apparent explanation is that Kaposi sarcoma is due to a transmitted agent different from HIV, the Kaposi sarcoma herpesvirus (KSHV) or HHV-8. Disease due to mycobacteria of the Mycobacterium avium–intracellulare complex is common, and patients with AIDS are also highly susceptible to Mycobacterium tuberculosis infection. Oral thrush and esophagitis due to Candida albicans and meningitis due to Cryptococcus are commonly encountered fungal infections. Persistent progressive mucocutaneous herpes simplex and herpes zoster infections are common. Cytomegalovirus (CMV) chorioretinitis is one of the most common opportunistic infections seen at very low CD4 T-cells count and may result in unilateral or bilateral blindness. Disseminated CMV infection is also seen, and patients present with fever and visceral (eg, gastrointestinal) organ involvement.

Specific opportunistic infections are associated with differing levels of CD4+ T-lymphocyte counts. For example, fungal and tuberculous pneumonia may occur with CD4+ T-lymphocyte counts of 200 to 500 cells/mm³, whereas CMV and M avium–intracellulare disease are seen almost exclusively in those whose counts are lower than 50 to 100 cells/mm³. Patients with opportunistic infections are treated for specific infections or conditions. However, with current ART regimens and patient management, the number of opportunistic infections has significantly reduced in the United States and other developed countries.

The CDC Classification of Clinical Categories of HIV-1 Disease that could be used to clinically categorize HIV-1 disease includes (1) A (asymptomatic, acute, HIV-1 or persistent generalized lymphadenopathy), (2) B (symptomatic conditions, some opportunistic infections, not A or C), and (3) C (AIDS indicator conditions). These categories are further subcategorized based on CD4 T-cell counts and clinical conditions described for each category including (1) CD4 T-cell count ≥ 500 could be A1, B1, or C1, (2) CD4 T-cell count 200-499 could be A2, B2, C2, and (3) CD4 T-cell count < 200 could be A3, B3, and C3. List of clinical conditions of each category could be seen at CDC website.

As the duration of survival of patients with AIDS became longer as a result of ART with the earliest drugs, an increased number of patients developed neurologic manifestations of the disease and lymphoid neoplasms, especially non-Hodgkin lymphomas. HIV is a neurotropic virus and can be isolated from the cerebrospinal fluid (CSF) of 50% to 70% of patients. CNS involvement may be asymptomatic, but many patients develop a subacute neurologic illness that produces clinical symptoms varying from mild cognitive dysfunction to severe dementia. Loss of complex cognitive function is usually the first sign of illness. Progression to severe memory loss, depression, seizures, and coma may ensue. Cerebral atrophy involving primarily cortical white matter can be demonstrated by computed tomography or magnetic resonance imaging. Histologically, focal vacuolation of the affected brain tissue with perivascular infiltration of macrophages is noted. Multinucleated giant cells with syncytium formation surround the perivascular infiltrates. Neurologic symptoms do not usually occur until CD4+ T-lymphocyte counts are lower than 200 cells/mm³.

The disease spectrum in Africa is similar in many respects to that in the Western world, but many more patients present with severe intractable wasting and diarrhea, known as slim disease. Tuberculosis is also more commonly encountered in AIDS patients in Africa, reflecting the higher incidence of the disease in the population in general. The 2-year mortality rate of persons with AIDS, once the disease has been fully established, was initially 75%, with nearly all persons eventually dying of opportunistic infections or neoplasms. However, with the accessibility of ART and other preventive measures, HIV-related deaths and new infections have declined in these countries.

DIAGNOSIS

The diagnosis of HIV infection can be done by three major types of available tests, including (1) antibody tests, (2) combination—4th-generation antibody–antigen tests, and (3) nucleic acid amplification tests based on PCR. Some of these tests differentiate between HIV-1 and HIV-2.

1. Antibody Test: The most commonly used HIV test has been by demonstrating antibody to HIV or its components (antigens). Initial screening tests are performed using whole viral lysates as the target antigens in enzyme-linked immunosorbent assay (ELISA) test. This test has a high level of sensitivity, but because false-positive results occur, all positive ELISA tests must be confirmed. The confirmatory test is a western blot analysis that detects antibodies to specific HIV-1 proteins. In this procedure, HIV-1 proteins are separated by electrophoresis, transferred to nitrocellulose paper, and incubated with patient sera; antibody bound to the individual proteins is detected by enzyme-labeled anti-human globulin sera (see Figure 4–15). Sera from infected patients have antibodies that react with the envelope glycoproteins, core proteins, or both. Tests performed for HIV-1 detect antibody in 60% to 90% of patients infected with HIV-2. The combination of ELISA and western blot tests gives a high degree of specificity to test results, but antibody is detectable by these procedures in the first 3 to 12 weeks after infection. This is called window period and 97% of infected people are likely to develop antibody during this period. If a suspected individual test is negative during window period, the test should be repeated 3 months after exposure. During this window period, the individual can still transmit the infection to others by sexual contact or blood donation. Therefore, nucleic acid test is performed for blood transfusion.

   Antibody-based ELISA tests were improved in their sensitivities and added HIV-2 antibody, in addition to HIV-1 antibody known as 2nd- and 3rd-generation HIV tests. In 3rd-generation test, both IgM and IgG antibodies were detected that improved the sensitivity. The FDA has also approved several rapid HIV antibody tests that can be performed in 30 minutes and used in both clinical and nonclinical settings, including home and can help to overcome some of the barriers to early diagnosis. These screening tests use oral swabs (saliva) or blood and are interpreted visually and require no instrumentation. Like the ELISA test, all require confirmation if reactive. In these rapid tests, HIV antigens are affixed to the test membrane and if HIV antibodies are present in the specimen being tested, they bind to the affixed antigen. The colorimetric reagent provided in the kit binds to these immunoglobulins and is visually detected.

2. Combination Antibody/Antigen-4th-Generation Test: This test detects HIV-1 and HIV-2 antibodies and HIV-1 antigens in blood within 2 to 6 weeks after infection. HIV virions appear in 1 to 4 weeks after infections, which means HIV antigens such as p24 (capsid protein) can be detected early in infection before the development of antibodies. Antibodies will be produced within a week after the appearance of antigens. This sensitive and specific test can detect both HIV antigen and antibody in 2 to 6 weeks after infection. Antigen and antibody detection in this combination utilizes ELISA-based technology. There are three steps involved in this test. If a patient tests positive in Step 1 it means HIV-1 p24 antigen is present, Step 2 differentiates between HIV-1 and HIV-2 antibodies, and if positive for either antibodies, confirms HIV-1 infection. However, if Step 2 is negative or indeterminate, Step 3, the nucleic acid test by PCR to detect HIV-1 RNA is performed, and if positive, confirms HIV-1 infection. If Step 3 is negative, then Step 1 was a false positive and result is HIV negative. The 4th-generation test is now widely used in the United States and many countries. There is a rapid version available that used blood or saliva samples.

3. Nucleic Acid Test: More practical approaches include nucleic acid-based assays such as the polymerase chain reaction (PCR) for plasma HIV-1 RNA (RT-PCR) or HIV-1 DNA (in peripheral blood mononuclear cells) and the branched-chain DNA (bDNA) assay. HIV-1 RNA in plasma of infected individuals can be detected 7 to 28 days after infection. These nucleic acid detection methods are also useful in assessing the benefits of antiviral therapy, as well as in determining whether infants born to seropositive mothers are infected or simply demonstrating passively transmitted transplacental antibody.

   Quantitation of plasma HIV RNA plays an especially important part in management. For example, if a patient’s HIV RNA copy number rises during therapy, or fails to fall to low levels (eg, lower than 50 copies/mL), this signals that the antiviral efficacy of the drug regimen is inadequate. The most likely explanation is mutational resistance that either preexisted or developed during treatment. Other explanations to be considered include patient noncompliance and inadequate dosing.

4. HIV diagnosis in infants born to infected mothers: Since maternal IgG is transferred to the fetus, antibody test will not diagnose HIV in infants. Therefore, HIV diagnosis in infants is confirmed by HIV-1 culture or HIV DNA/RNA PCR and positive results are confirmed repeating the test. HIV DNA PCR positivity is 38% for 48 hours of life, 93% for 14 days, and 98% for 4 weeks.

SCREENING

The CDC and US Preventive Task Force (USPTF) recommend that clinicians screen all adolescents and adults aged 13 to 64 years for HIV infection at least once as part of routine annual. The screening should be repeated annually for those who are at increased risk of HIV infection. In addition, MSM and bisexual men and those who inject drugs could benefit for more frequent testing like every 3 to 6 months. HIV screening should also be included in the routine panel of prenatal screening for all pregnant women. This recommendation is in line with the CDC guidelines of 2006 that HIV testing should be a part of routine healthcare for all adolescents and adults. It is believed that implementation of this recommendation may help 156 300 Americans (this number used to be 240 000 5 years ago), who are unaware of their HIV infection status, to learn their status and start receiving treatment. More than 30% of HIV infections are transmitted by those who are unaware of their HIV status. This recommendation has helped and will be helpful in further reducing HIV cases in the United States.

TREATMENT

Currently, there are six classes of antiretroviral agents, including nucleoside analog reverse transcriptase inhibitors (NRTIs), nonnucleoside analog reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), the gp41 fusion inhibitor, CCR5 antagonists, and integrase strand transfer inhibitors (INSTIs). These anti-HIV-1 agents are listed in Table 18–4. These inhibitors are used in a combination therapy (at least three separate inhibitors from two different classes) known as ART. In addition, in some combinations, a pharmacokinetic enhancer is added to increase its effectiveness of HIV-1 regimen.

The Department of Health and Human Services (HHS) has a working group (panel) for developing guidelines for antivirals use in adults and adolescents in the office of AIDS Research Advisory Council (OARAC). The current guidelines were updated on July 14, 2016 and are updated periodically (www.hhs.gov). The recommendation is to use ART for all HIV-infected individuals regardless of CD4 T-cell count to reduce morbidity and mortality associated with HIV infection. For treatment of naïve patients, ART generally consists of two nucleoside reverse transcriptase inhibitors (NRTIs) and a third antiviral that could be either an integrase strand transfer inhibitor (INSTI), a nonnucleoside reverse transcriptase inhibitor (NNRTI) or a protease inhibitor (PI) with a pharmacokinetic (PK) enhancer (booster) such as cobicistat or ritonavir.

The HHS panel recommended the following HIV regimens (recommended regimen) for ART in treatment-naïve patients. For INSTI-based regimens: two NRTIs (Tenofovir DF and Emtricitabine) plus an INSTI (Dolutegravir); or two NRTIs (Tenofovir AF and Emtricitabine) plus an INSTI (Elvitegravir) with a PK enhancer (Cobicistat); or two NRTIs (Tenofovir DF and Emtricitabine) plus an INSTI (Raltegravir). For HLA-B*5701 negative patients: two NRTIs (Abacavir and Lamivudine) plus one INSTI (Dolutegravir). For PI-based regimens: two NRTIs (Tenofovir DF and Emtricitabine) plus a PI (Darunavir) with a PK enhancer (Ritonavir). The alternative regimens are two NRTIs (in similar combinations listed earlier) plus one nonnucleoside reverse transcriptase inhibitor, NNRTI (Efavirenz or Rilpivirine). The alternate regimes are effective but have limitations for certain patient population. Several other combinations, alternative regimen options and other regimen options are available on HHS website. Several of these combinations are available in a single pill listed in Table 18–4. Within 6 weeks of ART, many patients see plasma HIV RNA reduction by more than 1 log, and by 6 months of treatment, HIV RNA should be almost undetectable (less than 50 copies of HIV-1 RNA/mL). With the suppression of HIV load, patients should see their CD4 T-cells count increasing. ART must be continued indefinitely to keep the viral load suppressed.

HIV regimen is also recommended for HIV-suspected occupational and nonoccupational postexposure prophylaxis (PEP). For PEP, INSTI-based regimen should be started as soon as possible but before 72 hours post exposure, and should be given for 28 days followed by regular testing for HIV by 4th-generation HIV test. ART has also shown to prevent HIV transmission and can be used as preexposure prophylaxis (PrEP) by HIV negative partners of HIV-positive partners. For PrEP, the FDA approved daily dose of two NRTIs, tenofovir and emtricitabine (Truvada), in combination with safer sex practices, can reduce the risk if HIV-1 transmission by 90% from sex and 70% from injection drug use. For prevention of mother-to-child transmission during pregnancy, the preferred regimens in treatment-naïve pregnant women are: two NRTIs (Abacavir and Lamivudine or Emtricitabine and Tenofovir or Tenofovir and Lamivudine) plus one PI (Atazanavir or Darunavir) or one INSTI (Raltegravir). The use of ART during pregnancy has reduced the mother-to-child transmission rates by less than 1% in the United States.

Recent advances in HIV therapy have slowed progression of the HIV disease and appears to be responsible for dramatic improvement in many patients lives, but toxicity or the development of resistance remains the concern. However, successful suppression of HIV by ART can reconstitute CD4 T-lymphocyte numbers that cause an inflammatory response known as immune reconstitution inflammatory syndrome (IRIS). Some of the common coinfections that may be exacerbated by IRIS are tuberculous and nontuberculous mycobacteria, CMV retinitis, cryptococcal meningitis, hepatitis B, and hepatitis C. In addition to side effects of antiretrovirals, several complications of ART include lipoatrophy (visceral fat accumulation), hypercholesterolemia, low HDL, hypertriglyceridemia, insulin resistance, impaired glucose tolerance, cardiovascular disease, lactic acidosis, osteopenia, osteoporosis, osteonecrosis, and others.

Initiation of Treatment

Because HIV replication proceeds at such a phenomenal rate, it seems most rational to begin treatment as soon as infection is detected. Therefore, ART is recommended by HHS panel for all HIV-infected individuals regardless of CD4 T-cell count to reduce the morbidity and mortality related to HIV infection. ART is also recommended to prevent adult HIV transmission and mother-to child transmission. In some instances, ART may be deferred because of clinical and/or psychological factors, but should be started as soon as possible. In addition, several conditions increase the urgency to start ART, including pregnancy of HIV-infected women, AIDS-defining illness, acute opportunistic infections and malignancies, CD4 T-cell counts of lower than 200 cells/mm³, HIV-associated nephropathy, acute HIV infections (acute retroviral syndrome), coinfection with HBV or HCV. However, considerations of toxicity, resistance development, quality of life, cost, and patient wishes are extremely important additional determinants. Before the initiation of ART, plasma HIV RNA (viral load), CD4 T-cell count, HIV genotyping (to determine ART resistant mutants), and other laboratory parameters should be performed. The efficacy of ART should be followed by performing viral load and CD4 T-cell count and the adverse effects of the ART should also be evaluated. Because current therapy is unlikely to eradicate HIV infection, most patients are likely to stay on therapy for life.

Resistance

HIV error-prone reverse transcriptase enzyme and high rates of viral replication contribute to frequent mutations. As a result, resistance to an antiviral is a regular and often rapid development. Use of antiviral therapies that maximally suppress HIV viral load appears to diminish the appearance of resistant virus, especially combination therapy. The emergence of resistance occurs at a rate proportional to the frequency of preexisting variants and their relative growth benefit in the presence of antiviral. Antiviral resistance is determined before the start of therapy and during the therapy if viral suppression is not achieved. In addition to the primary antiviral treatment of HIV, patients with CD4+ counts of less than 200/mm³ should begin prophylactic regimens to prevent P jiroveci pneumonia. When CD4+ counts are less than 75 to 100/mm³, they should receive prophylaxis for mycobacterial and fungal infection.

PREVENTION

The spread of AIDS has been facilitated by changing sexual mores, injection drug use, and, in some parts of the world, disruption of family and tribal units because of industrialization and urbanization. Immediate prevention must be based on education about the means of transmission and easy access to condoms and safe needles for those large numbers of people who continue to place themselves at risk. ART is recommended to prevent HIV transmission in high-risk groups. Apart from questions of potential discrimination against infected individuals, there are calamitous effects of false-positive serologic test results. However, next-generation HIV tests have improved the sensitivity and specificity to avoid false laboratory results.

Much research is underway to develop vaccines against the virus, but the marked mutability of HIV greatly complicates this approach. Furthermore, passage of virus between fused cells and in syncytia protects it from antibody neutralization in established disease. The search continues for conserved epitopes of the surface glycopeptides that might provide possible antigenic targets. Antiviral treatment using combinations of agents may prevent infection of accidentally exposed individuals (eg, health-care workers). This therapy must be initiated within hours of an accident if it is to have any chance of success. Detection and treatment of HIV-infected pregnant women is very effective in reducing perinatal infection. Cesarean section delivery, particularly that which is elective rather than emergent, is also preventive, as is the avoidance of breastfeeding by HIV-positive mothers. Latex condoms, properly used, do prevent HIV transmission bidirectionally, and with efficacy rates up to 98% to 99%. Circumcision of males decreases the risk of acquisition of HIV by 60% in men, but has not been clearly shown to reduce transmission to females. Screening of blood for HIV by nucleic acid testing by PCR is very effective.

Human T-lymphotropic virus or human T-cell leukemia virus (HTLV) has two members, HTLV-I and HTLV-II, which cause disease in humans. HTLV-I causes two distinct diseases; adult T-cell leukemia/lymphoma (ATLL) and HTLV-associated myelopathy (HAM, a neurologic disease). HTLV-II may also cause these diseases but has been primarily linked to variant hairy cell leukemia.

VIROLOGY

Similar to other retroviruses, HTLV has the usual retroviral gag, pol, and env genes, but also encode two regulatory proteins: Tax and Rex. Tax is a transcriptional activator of HTLV LTR and is also required for transformation. However, Rex, similar to HIV-1 Rev, is a posttranscriptional activator that increases transport of structural protein mRNAs from nucleus to cytoplasm. In addition, other HTLV proteins are similar to HIV-1 proteins but differ in sequence and antigenicity. The HTLV envelope glycoproteins are gp46 and gp21, whereas the capsid protein is p24. Several cellular factors interact with HTLV LTR and activate transcription. Unlike HIV-1, the receptors for HTLV-I and HTLV-II have not been biochemically identified. However, the receptors are found in a wide variety of human and animal cells. HTLV-I and HTLV-II use the same receptor. HTLV is able to penetrate and infect a number of cell types; however, productive infection is observed in only a few cell types such as T-lymphocytes. The replication cycle of HTLV is very similar to that of HIV-1. Syncytia formation has been demonstrated in T-lymphocytes.

TRANSMISSION

Transmission of HTLV occurs via blood to blood, including anal and vaginal sex and intravenous drug use. Mother-to-child transmission of HTLV has also been documented. Unlike HIV-1, HTLV is not transmitted through cell-free fluids, but through cell-associated fluids.

EPIDEMIOLOGY

HTLV is more prevalent in the Caribbean, Japan, and Hawaii. In addition, the incidence of HTLV is increasing in Western Europe and the United States among intravenous drug users. In some of these endemic areas, the rate of HTLV infection is more than 20%.

PATHOGENESIS

Adult T-cell leukemia/lymphoma is caused by HTLV-I infection of CD4 T-lymphocytes leading to malignant transformation. HTLV-encoded Tax protein that binds to HTLV LTR and increases transcription of HTLV genes is also responsible for enhancing the transcription of protooncogenes resulting in transformation (see later under “Transformation by animal and human oncoretroviruses” section). In addition, Tax increases the production of IL-2 (T-cell growth factor) and IL-2 receptor that cause uncontrolled growth of T cells resulting in transformation. The transformed cells typically do not produce HTLV progeny viruses. The other disease caused by HTLV is called HAM, or tropical spastic paraparesis (TSP), which is a demyelinating disease of the brain and spinal cord, especially the motor neurons. It is believed that the mechanisms of HAM/TSP are immune mediated, including an autoimmune reaction-induced damage of the neurons as well as cytotoxic T-cell–induced killing of neurons. The virus becomes latent for a long period of time (approximately 20-30 years) or slowly replicates to transform cells without causing cytopathic effects. In terms of immunity, antibodies are elicited against gp46 and other HTLV proteins that neutralize the slowly replicating virus and prevent cell-mediated killing of HTLV-infected cells.

MANIFESTATIONS

HTLV-I causes ATLL, which is a highly malignant disease. There is a long period of latency (about 20-30 years) before onset of ATLL. Only 1% to 2.5% of infected people progress to ATLL disease, and their survival is often in months. ATLL patients present with lymphadenopathy, hepatosplenomegaly, and skin and bone lesions. The malignant T cells have a flower-shaped nucleus and are pleomorphic. Fungal and viral opportunistic infections are commonly seen in ATLL patients, especially those treated with aggressive chemotherapy. In HAM/TSP patients, gait stiffness/spasticity, lower limb weakness, and low back pain are generally seen. The flower-shaped T cells can be found in the CSF. The CSF shows lymphocytic pleocytosis, and the protein level is elevated. In addition, hematologic malignancies, B-cell chronic lymphocytic leukemia, and immunosuppression are found in patients infected with HTLV-I. HTLV-II causes a T-cell variant hairy cell leukemia, which resembles hairy cell leukemia of B-cell origin.

DIAGNOSIS

HTLV infection is diagnosed by detection of antibodies against HTLV by ELISA; however, there is cross-reactivity with HTLV-I and HTLV-II antigens. PCR can specifically differentiate between HTLV-I and HTLV-II. ATLL is diagnosed by the presence of malignant T cells in the lesions. HAM/TSP is diagnosed by the presence of HTLV antibody in the CSF or HTLV nucleic acid in the CSF.

TREATMENT

In some patients with HAM/TSP, a combination of antiretrovirals and interferon has shown benefit, and corticosteroids may relieve symptoms. ATLL is generally treated by anticancer chemotherapy.

PREVENTION

Screening for HTLV antibodies, using condoms, and not breastfeeding babies by HTLV-infected mothers can reduce the risk of HTLV transmission. Currently, there is no vaccine to prevent HTLV infection.

TRANSFORMATION BY ANIMAL AND HUMAN ONCORETROVIRUSES

Oncoretroviruses cause a variety of cancers in animals and humans, including leukemia, lymphoma, and sarcoma. Oncogenic retroviruses appear to transform cells to an oncogenic state by three distinct mechanisms: By acquiring a cellular oncogene (acute transforming animal retroviruses), by insertional mutagenesis (animal retrovirus), and by transforming cells by continual expression of viral regulatory Tax protein for human retrovirus, HTLV (see Chapter 7). The genomes of acute transforming oncoviruses have one feature common to nearly all of them: Some viral genes are replaced by host genes derived from their hosts that render them oncogenic (see later in text). In every case, the signals required for reverse transcription and for transcription of the provirus, which are located near the ends of the RNA, are retained in the infecting virus. In the example shown in Figure 18–6, the pol gene and parts of both the viral gag and env genes are deleted, but other configurations are possible. Such oncoviruses are defective and replicate only in the presence of a helper virus that can supply the missing functions.

First, the defective acute transforming viruses (Figure 18–6) have acquired a cellular gene (thereafter called an oncogene), which, when expressed in the infected cell, results in loss of normal growth control. On infection, the transduced oncogene is expressed from the viral LTR promoter, resulting in a rapid and acute onset of malignant disease. Persistent transformation by oncogene transduction is possible only for retroviruses that are not cytocidal. More than 30 oncogenes have been identified in a variety of animal retroviruses, but no human retroviruses are known that transform by this mechanism.

The second mechanism is called insertional mutagenesis. Integration of an animal retrovirus in the vicinity of particular cellular genes can cause inappropriate expression of the gene, resulting in uncontrolled cell growth. These cellular genes are called protooncogenes, and insertional activation by the virus is apparently due to the close proximity of the integrated viral promoter or enhancer to the gene. Cancers that are caused by this mechanism have very long latent periods, because integration is random and only rarely occurs near a cellular protooncogene in case of animal retrovirus infection.

The causative agent of ATLL, HTLV-I, exemplifies the third mechanism. In this case, the integrated provirus in the leukemic cells from any one patient is found at a unique location on a particular chromosome. Thus, the tumors are probably monoclonal. The cancer is not the result of insertional activation, however, because the chromosomal location of the provirus is never the same in any two patients. Instead, transformation results from the continual expression of the viral tax gene (the HTLV-I homolog of the HIV tat gene; Table 18–2). Apparently, the Tax protein not only can transactivate viral transcription in the same manner as HIV Tat, but Tax can also transactivate the expression of one or more cellular genes (possibly protooncogenes), resulting in malignant transformation.