21

CHAPTER

H um an Im m unodef ci i ency V i rus

(A cqui Im m unodef ci red i ency Syndrom e)
(Dr. R Chawla chawlarn2@yahoo.com) OBJECTIVES 1. To study the spectrum of human immunodeficiency virus pandemic. 2. To learn the properties of lentiviruses. 3. To study the molecular structure of HIV. 4. To know the life-cycle and pathogenesis of the virus. 5. To learn about the diagnostic tests available for HIV testing. 6. To study the therapeutic approaches to control HIV infection. 7. To understand various approaches to the development of a vaccine against HIV. Approximately 1 in every 100 adults world wide (more than 50 million people) between the age of 15 and 49 is infected with HIV, the human immunodeficiency virus. The syndrome first detected in the year 1981 in Gay population in United States of America and labeled as Gay Plague, is not restricted to the homosexuals. More than 75% of all adult HIV infections have resulted from heterosexual intercourse. Each day, about 16,000 people worldwide are infected with the virus, with 95% of new cases occurring in developing countries. Women and children appear to be more prone to the infection. About 43% of adults living with AIDS worldwide are women, and this proportion is growing. Out of more than 2.6 million people who died of AIDS-associated diseases in 2007, an estimated 550,000 were children under 15 years of age. Mother-to-child transmission is responsible for more than 90% of all HIV infections in infants and children around the world. The acquired immunodeficiency syndrome (AIDS) was first detected by Michael Gottlieb in Los Angeles, USA in 1981, when five cases of opportunistic infections, pneumocystitis carinii pneumonia were reported. At the time, when even a single case with this infection could raise an alarm in the medical field, reporting of five cases was indication of an epidemic. Investigations revealed that the individuals were immunodeficient and had low CD4 + lymphocyte counts. The individuals were found to be homosexuals and hence, the disease was labeled as Gay related immunodeficient disease (GRID). In 1983, first series of deaths due to AIDS was reported from Kinshasa district in Zaire and heterosexual transmission of the virus was confirmed. The world was shocked but helplessly, entered into the HIV Era. INDIAN SCENARIO The first AIDS case in India was detected in 1986; since then HIV infection has been reported in all States and Union Territories. The spread of HIV in India has been diverse, with much of India having a low rate of infection

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and the epidemic being most extreme in the southern half of the country and in the far northeast. The highest HIV prevalence rates are found in Maharashtra, Andhra Pradesh, Karnataka, Manipur and Nagaland in the North-East. As of August 2006, the greatest numbers were in Maharashtra and Gujarat in the West; Tamil Nadu, Andhra Pradesh and Karnataka in the South; and Manipur and West Bengal in the North-East. In the southern states, the infections are mostly due to heterosexual contact, while infections are mainly found amongst injecting drug users in Manipur and Nagaland. Previously it was thought that around 5 million people were living with HIV in Indiamore than in any other country. Better data, including the results of a national household survey, led to a major revision of the prevalence estimate in July 2007. As per these statistics, people living with HIV/ AIDS in India is about 2 million and adult (15 years and above) HIV prevalence is 0.36%. THE RETROVIRUS Retroviruses are the viruses that carry their genome in the form of ribonucleic acid (RNA). The genomic RNA has to be replicated to deoxynucleic acid (DNA) before it can interact and intercalate with the host cell DNA. The enzyme responsible for this conversion i.e. RNA to DNA is called Reverse Transcriptase since it reverses the central dogma of flow of information from DNA to RNA to proteins (Fig. 21.1). The retroviruses are a large and diverse family of RNA viruses characterized by the intermediate existence of double stranded linear DNA that intercalates into the host genome providing a period of latency in the life cycle of the virus. The first retroviruses discovered in 1979 and 1981 were called human T-cell leukemia/lymphoma virus (HTLV) type I and type II. In 1983, Barre-Sinoussi and co-workers from Pasteur Institute recovered a Reverse Transcriptase containing virus from the lymph nodes of a patient with lymphadenopathy syndrome. This virus, however, proved to be different from HTLV as it killed CD4+ cells rather than their propagation in cell cultures. JA Levy (1984) was the first to isolate the retrovirus from

Flow of information

DNA

RNA
Reverse Transcriptase

DNA mRNA
Integrase

Viral DNA integrated into Host Genome

mRNA Proteins central dogma

Proteins

Fig. 21.1: Flow of genetic information in retroviruses

an AIDS patient and called it AIDS associated virus (ARV). Subsequently, it was established that the genome and the proteins of ARV were distinct from HTLV and hence International Committee of Taxonomy of Viruses recommended a separate name for ARV i.e. Human Immunodeficiency Virus. The infectious retroviruses have further been classified into seven genera. The human retroviruses include lentiviruses (HIV-1 and HIV2), onc viruses (HTLV-I and HTLV-II) and human endogenous virus (HERV-K). All the human retroviruses show T-cell tropism, genomic complexity and mutation capability. The high mutation rate provides the viruses with a great survival benefit and is, at least partly, attributed to the error prone reverse transcription by the RNA dependent DNA polymerase (reverse transcriptase) which lacks a proofreading step during the polymerization. The primate lentiviruses: As mentioned above, HIV represents the genus lentivirus. The characteristics of these viruses include: a. They can efficiently infect terminallydifferentiated, non-dividing cells such as macrophages. Tissue resident macrophages, such as brain macrophages (or microglia) may be important for development of specific aspects of virally-induced disease.

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b. In addition to structural (gag, env) and enzymatic (pol) proteins, their genome also encodes regulatory proteins. Most notable among these are Tat-like and Rev-like proteins which, respectively, regulate viral transcription and viral RNA transport. c. In terms of the pathogenesis of lentivirus infections, some key properties are as follows: i. Lentiviruses persist life-long. ii. Lentiviruses have high mutation rates. iii. Infection proceeds through at least three stages viz. infection, latency and lysogeni. iv. Onset as well as presentation of the disease varies widely between individuals. Phylogenetic studies of primate lentiviruses have provided compelling evidence that HIV-1 is closely related to a virus that naturally infects chimpanzees (SIVcpz), whereas HIV-2 is closely related to a virus that naturally infects sooty mangabey monkeys (SIVsmm). It is believed that
Table 21.1: Properties of lentiviruses Family Human viruses Particle size Envelope Shape of capsid Genome Size of Genome Complications Retroviridae HIV-1, HIV-2, HTLV-I and HTLV-II 80 130 nm Present Icosahedral Diploid linear, Sense, single stranded RNA 10 kb Immunodeficiency, Opportunistic infections (tuberculosis and pneumonia), Neurologic, Arthritis, Kaposis sarcoma Variable (2-10 years)

are resident in the same region of West Africa in which HIV-2 is most prevalent. Thus, HIV-1 and HIV-2 represent emerging infections developed by interspecies cross infection from monkeys to man. Of the two viruses associated with human AIDS, HIV-1 is distributed throughout the world i.e. the global epidemic (pandemic) is due to HIV-1. HIV2 appears to be less pathogenic and remains largely restricted to West Africa. The epidemiological evidence indicates that HIV-2 may be less pathogenic than HIV-1 and that the HIV-2 infected persons may be at a decreased risk for acquiring HIV-1 infection. Clinical Features of HIV Infection The disease progression in HIV infected individuals can be divided into three distinct stages: 1. Acute primary infection syndrome: The primary HIV infection can be asymptomatic, or it may be associated with an influenzalike illness with fevers, malaise, diarrhea and neurologic symptoms such as headache. This illness usually lasts 2 to 3 weeks, with full recovery. The symptoms are so subtle and common that it is difficult to relate them to HIV infection unless the individual is aware of the exposure. 2. Asymptomatic infection: This refers to the asymptomatic carrier state that follows initial infection. Although silent, the disease is progressing during this period as more and more T-cells are being infected and lysed. It typically lasts for many years, with a gradual decline in the number of circulating CD4+ Tcells. During this period the frequency of common infections might increase. In a minority of cases, infection does not proceed beyond this asymptomatic phase and CD4 + counts remain stable (these persons are known as long-term survivors or long-term non-progressors). 3. Symptomatic HIV infection and AIDS: Symptoms that are related to HIV infection

Disease Progression

zoonotic ( trans-species) transfer of these simian immunodeficiency viruses resulted in the emergence of HIV-1 and HIV-2. Sooty mangabeys

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ultimately begin to develop as a result of continuously decreasing CD4+ cell counts and the associated immunodeficiency. This last fatal phase of the HIV infection is known as acute Immunodeficiency syndrome (AIDS), is defined by more serious AIDS-defining illnesses and/or by a decline in the circulating CD4+ count to below 200 cells per microliter. The opportunistic infections like pneumo cystic carinii pneumonia and mycobacterium tuberculosis take charge, skin and intestinal infestations become common occurrence. Toxo-plasmosis of the brain, CMV (cytomegalovirus) retinitis and malignancies like cervical cancer, Kaposis sarcoma and various B-cell lymphomas linked to EBV further complicate the condition. The other AIDS related illnesses include: HIV-related encephalopathy, HIVrelated wasting syndrome and lymphoid interstitial pneumonia (kids). AIDS typically occurs about 8 to 12 years after initial HIV-1 infection. Structure of HIV HIV virion has a fairly complex structure. Microscopic examination shows a roughly spherical particle but the capsid layer has an icosahedral structure. Nucleic acid: The virus is thought to contain two identical copies of a positive sense singlestranded RNA molecule, about 9,500 nucleotides long. The two RNA molecules might be linked to each other to form a genomic RNA dimer. The RNA strands are associated with a basic nucleocapsid (NC) protein (p9). In case of other RNA viruses, the nucleoprotein filament is helical, but in HIV the helicity has not been confirmed. Capsid: The ribonucleoprotein particle is encapsidated by a capsid made up of a capsid protein (CA), p24. The capsid environment also contains other viral proteins such as integrase and reverse transcriptase (Figure 21.2) along with a wide variety of other macromolecules derived from the cell. An intracapsid molecule tRNAlys3 serves as a primer during reverse transcription. The capsid has an icosahedral structure. Envelope: The capsid is in turn encapsidated by a layer of matrix protein (MA), p17. This matrix protein is associated with a lipid bilayer or envelope. In HIV, the matrix protein makes a continuous shell attached to the envelope. The HIV envelope is derived from the host cell plasma membrane and is acquired when the virus buds through the cell membrane. The viral envelope contains the lipid and protein constituents of the membrane from which it is derived. In addition, it also contains viral proteins often forming spikes or peplomers. The major HIV protein associated with the envelope is gp120/41. This functions as the viral antireceptor or attachment protein. gp41 is a transmembrane protein as it traverses the envelope. On the other hand, gp120 is present on the surface and is noncovalently attached to gp41. Both of these proteins are synthesized in the endoplasmic reticulum as a large protein (gp160), which is glycosylated and split into the gp120 and gp41 proteins, in Golgis apparatus. A hydrolytic enzyme protein, protease, is present inside as well as outside the capsid. An envelope is a common feature in animal viruses but uncommon in plant viruses. In the case of Herpes viruses, the envelope is derived from the nuclear membrane. On the other hand, viruses such as vaccinia derive an envelope from the Golgi body. HIV GENOME The HIV genome is 9749 nucleotidesabout the same size as other retroviruses like Rous Sarcoma Virus (RSV) but the genome of HIV is more complex than that of RSV. It has extra open reading frames that code for small proteins. Antibodies against these small proteins are found in HIV-infected people. Both the ends of the genome carry long terminal repeat sequences (LTRs).

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rp ot e ro i n te s in s

P6 P9

(MA) P17 (CA) P24

Gp120

Gp41

Integrase Viral RNA Reserve transcriptase Protease Host cell proteins

Pol pro teins

Fig. 21.2: Structure of human immunodeficiency virus (HIV-1)

The HIV genome has nine open reading frames (leading to nine primary translation products) but 15 proteins are made in all as a result of cleavage of three of the primary products. The gag and pol genes together are translated into large polyproteins, which are then cleaved by a virusencoded protease that is part of the pol polyprotein. Pol gene: The pol gene codes for the viral enzyme proteins i.e. reverse transcriptase (RT), protease (PR) and integrase (IN). As described above, reverse transcriptase is the most important enzyme as it translates RNA into the double stranded DNA. Integrase helps in the intercalation of double stranded DNA into the host genome. Protease is the processing enzyme which cleaves different gene products into the mature viral proteins.

Gag gene: The gag gene codes for a polyprotein p55 which is cleaved into four proteins that are found in the mature virus: MA (matrix), CA (capsid), NC (nucleocapsid) and p6. Env gene: env stands for the envelope and hence the env gene codes for the envelope proteins. env gene is translated to a polyprotein (Gp160) which is then cleaved by a host cell protease (called furin) found in the Golgi body. It is not cleaved by the virus-encoded protease. Gp160 is cleaved to: SU (Gp120) and TM (Gp41). The two proteins act as antireceptors and are involved in the interaction of the virion with CD4+ cells. The synthesis of gp160 takes place in endoplasmic reticulum and glycosylation in Golgis apparatus.

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Viral coat proteins (env products)

ag ga gp

( NC)

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In addition to the nine proteins derived from gag, pol and env, there are six other proteins made by HIV. Three of these are incorporated into the virus (Vif, Vpr and Nef), while the others are not found in the mature virus: Tat and Rev are small regulatory proteins and Vpu indirectly assists in assembly. They overlap with the structural genes (especially env) but are in different reading frames. From the diagram of the organization of HIV genome (Figure 21.3), it can be seen that some of the proteins are encoded in two exons (unlike the structural genes) and therefore their mRNAs can be derived by alternative splicing of structural gene mRNAs. Mutants in the TAT and REV genes show that both proteins are necessary for virus production. Functions of these regulatory proteins are described in brief. TAT (Trans-Activator of Transcription): TAT gene product binds to a sequence in all of the genes of HIV known as TAT responsive elements and positively stimulates transcription. It is thus a positive regulator of protein synthesis, including its own synthesis. REV (Regulator of Virion protein expression): REV binds to an element only in the mRNA for structural proteins (gag/pol/env) and regulates
p7, p9, p17, p24 (Nucleocapsid core proteins) Promotes infectivity

the ratio of gag/pol/env to non-structural, controlling protein (TAT/REV) synthesis. When REV levels are high, the rate of structural protein synthesis rises and that of controlling protein synthesis falls. Thus, REV inhibits its own production as well as that of TAT. The normal result is homeostasis, low or non-existent virus production and latency in the resting CD4+ cells. HIV uses genomic RNA as its messenger RNA, which is unspliced and hence cannot leave the nucleus. Rev functions to help in the transport of this mRNA out of the nucleus. Since the translation of this mRNA produces the gag and pol gene products, no viral proteins are synthesized when the rev gene is mutated or deleted. NEF (Negative Regulatory Factor): NEF is a small protein, approximately 27 kDa, synthesized early in infection. Despite its small size, NEF has several functions. a. The translation of the NEF gene as a result of the first infecting virus causes the internalization of CD4+ antigen from the cell surface and its destruction in lysosomes. Thus, no more HIV or gp120 can bind to the surface of an infected cell.
Structural gene expression Transcriptional activator

5 LTR U3 R U5

gag pol

vif vpr

rev tat

nef
3 LTR

env vpu

TAR element

Binding sites for host transcription factors

Gp120, gp41 viral coat proteins Reverse transcriptase, protease and integrase

Fig. 21.3: Schematic representation of the HIV genome and the proteins coded by different genes

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b. NEF also reduces the surface expression of MHC class I molecules. The infected cell is not able to present the viral proteins on its surface and hence is protected from attack by cytotoxic T cells. c. The virus produced in the presence of NEF is a little more infectious than the one produced in its absence. d. NEF increases the secretion of chemokines MIP-1a and MIP-1b from the infected macrophages. The chemokines cause the resting CD4+ T cells to migrate (undergo chemotaxis) towards the infected macrophages. Migration of uninfected cells towards infected cells increases the probability that the T cells will encounter infected macrophages before they leave the reticuloendothelial system. VPU (Viral Protein U): The small HIV protein, VPU enhances the pathogenicity of the virus by increasing the number of HIV particles per cell. It does that by means of two effects: a. It promotes the proteolysis of the CD4 antigen of host cell as it is made so that they should not bind to the Gp120 being made in the endoplasmic reticulum of the same cell. b. VPU also enhances viral particle release from the host cell. VPU also binds to a cellular protein (VPUbinding protein or UBP) and overexpression of this protein diminishes the enhancing effect of VPU on virus release. UBP may be a negative factor for assembly that must be displaced from one of the gag proteins before virus can assemble at the cell surface. VIF (viral infectivity factor) protein: VIF is essential for infection in vivo and may be very important in suppressing resistance to HIV infection by the host. It is needed during late stages of virus production and seems to function by suppressing innate anti-viral activities in T cells and macrophages. Without VIF, HIV is not infectious in primary human T cells. The mechanism appears to be the prevention of editing of the single strand form of viral DNA by the enzyme cytidine deaminase. The editing of DNA would lead to changes in the viral proteins and result in loss of viral infectivity. VPR (Viral Protein R): It influences the pathogenesis of HIV and is essential for infection of macrophages, and to a lesser extent, of other cells. It also activates HIV LTR-promoted transcription. It causes the arrest of host cell division in G2 stage of the cell cycle and apoptosis of the infected cell. It acts as a cytoplasmicnucleus shuttle protein (for the pre-integration complex through the nuclear pores). VPR is found in the serum of HIV-infected patients. Life-Cycle of the Virus The life-cycle of HIV-1 can be discussed under the following headings: a. Viral Infection (attachment and entry). b. Reverse transcription. c. Integration into the host genome. d. Transcription. e. Assembly. f. Budding. a. Viral infection: The life-cycle of HIV begins when a virion gets attached to a T lymphocyte or a macrophage. The high affinity binding takes place through the Gp120 envelope protein attachment with a CD4 molecule that acts as a receptor of the HIV virus. The CD4 molecule is a 55 kD protein expressed on the surface of T-helper and Tinducer lymphocytes as well as on the monocytes/macrophages and dendritic cells. The CD4 acts as a primary binder but a coreceptor is also required for the entry of virus into the cell. The co-receptor can either be a CCR5 or CXCR4 receptor, which is seven-transmembranedomain G-protein, coupled cellular receptor (Figure 21.4). After the virion has bound to the T-cell, the envelope undergoes conformational changes that facilitate the fusion of the

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Attachment of Gp120 with CD4 antigen CD4+ cell CD4 Antigen HIV ENTRY INTO A CD4 + CELL

Recruitment of Coreceptor

Coreceptor Exposure of Gp41 and membrane fusion

Capsid insertion into the T-cell

Fig. 21.4: Entry of HIV into the CD4+ T lymphocytes

envelope with the T-cell membrane. The fusion takes place in a coiled-spring fashion via a newly exposed Gp41 molecule which embeds in the membrane of CD4+ cells. Fusion of the membranes pushes the nucleocapsid into the T-cell. The nuclear targeting signal of the capsid then interacts with the nuclear membrane, probably at the nuclear membrane pore. The capsid membrane fuses with the nuclear membrane and the capsid contents are poured into the nucleus. b. Reverse transcription: The components of the HIV capsid released into the nucleus include the enzymes reverse transcriptase, integrase and protease along with the viral genomic RNA. Reverse transcriptase then polymerizes deoxyribonucleotides using viral RNA as the template to generate the double stranded DNA molecules. c. Integration: The newly formed HIV DNA in the host nucleus intercalates randomly at

multiple sites in the host genomic DNA. This integration of the viral DNA into that of host is achieved by an HIV enzyme called integrase. The integrated HIV DNA is called provirus. The provirus may remain inactive for several years, producing few or no new copies of HIV. This dormancy of the virus in resting memory T cells is referred to as cellular latency and may last for a few hours or days or very much longer in a very small minority of cells. Recently, it has been shown that the use of highly active anti-retroviral therapies (HAART) could eliminate the virus from the patient altogether but this minority of resting cells may provide a reservoir of integrated virus that cannot be eliminated by chemotherapy and may persist for a lifetime. Most viruses that replicate in the nucleus can do so only in dividing cells but cell division is not essential for HIV replication. This is because two viral proteins (Vpr and

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one of the GAG proteins) have nuclear localization signals and so nuclear membrane breakdown (mitosis) is not necessary. Cellular latency is a different phenomenon from clinical latency which refers to the fact that symptoms of HIV infection do not manifest themselves as AIDS for many years. Escape from latency: When the CD4 cell is stimulated during an antigenic response, the virus starts to proliferate and the latency is broken. A transcription factor i.e. nuclear factor NF kappa B (NFkB) appears to be involved in the process. The signal to grow is relayed from the cell surface to the nucleus by signaling proteins including transcription factors like NFkB. The NFkB binds to a site in the promoter region of the genes (particularly in the long terminal repeat regions of viral DNA) and stimulates its transcription. d. Transcription: When the host cell receives a signal to become active, the provirus uses the host RNA polymerase to create copies of the HIV genomic material, as well as shorter strands of RNA called messenger RNA (mRNA). The mRNA is used as a blueprint to make long chains of HIV proteins. Viral proteins are synthesized on the ribosomes and are glycosylated in the Golgis apparatus in the host cell. e. Assembly: The HIV protease cuts the long chains of HIV proteins into smaller individual proteins. As the smaller HIV proteins come together with copies of HIVs RNA genetic material, a new virus particle is assembled. f. Budding: The newly assembled virus pushes out (buds) from the host cell. During budding, the new virus envelope is formed partly, from the T-cell membrane and hence carries a number of host cell proteins along with the viral envelop proteins. The new copies of HIV are then free to infect other cells. The complete life-cycle of HIV is represented schematically in Figure 21.5.

Attachment
Reverse Transcription

Fusion

Entry DsDNA

Genomic DNA

Intercalation Viral RNA

Protein synthesis

Assembly Exit

Fig. 21.5: Life-cycle of human immunodeficiency virus in CD4+ T helper cells

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Modes of Transmission The major modes of transmission of HIV are: Unprotected penetrative sexual intercourse with an infected person. Infected blood and tissues: Donations of semen by infected person (artificial insemination). Skin grafts or organ transplants taken from an infected person. Transfusion of contaminated blood or blood products. From a infected mother to her baby; this can occur during: Pregnancy. Delivery; and Breastfeeding. Sharing unsterilised injection equipment that has previously been used by an infected person. In India, like in most other countries, the major route of HIV transmission is the unprotected sex (Table 21.2), followed by mother to child transfer. Stricter regulations for the blood banks, where all the donated blood bottles are mandatory to be tested for HIV, have reduced the transmission through donation of the infected blood.
Table 21.2: Proportion of different modes of transmission of HIV in reported cases (July 2007) Transmission Sexual Mother to child Injecting drug users Blood and blood products Unspecified Number of cases 106,669 4,755 2,930 2,563 8,078 Per cent 85% 4% 2% 2% 6%

coughing and sharing cutlery does not result in the virus being passed from one person to another.
Table 21.3: Relative risk associated with different modes of transmission of HIV Transmission Sexual Activity Unprotected sex Anal sex Oral sex Open mouth kissing Lesbian sex Kissing and other physical contact Infected Tissues Organ transplant Skin Blood Occupational Risk* Needle prick injuries Blood splashes Contaminated waste Mother to Child Gestation Delivery Breast feeding Moderate High Moderate Low Very low Extremely low Very high Very high Very high Very high Very high Low Low Low Extremely low HIV Risk

The HIV risk associated with different modes of transmission is tabulated in Table 21.3. HIV is not an airborne, water-borne or food-borne virus, and does not survive for very long outside the human body. Therefore ordinary social contact such as kissing, shaking hands,

All health care personnel are required to follow the universal precautions for infection control. The risk to healthcare workers being exposed to HIV is extremely low, especially if they follow universal healthcare precautions. Everyday casual contact does not expose anyone, including healthcare workers, to HIV. The main risk is through accidental injuries from needles and other sharp objects that may be contaminated with HIV. It has been estimated that the risk of infection from a needlestick injury is less than 1 percent. In the UK for instance, there have been five

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documented cases of HIV transmission through occupational exposure in the healthcare setting, and twelve possible/probable cases. In the US, there have been more than 50 documented cases of occupational HIV transmission. The risk posed by a needlestick injury may be higher if it is a deep injury; if it is made with a hollow bore needle; if the source patient has a high viral load; or if the sharp instrument is visibly contaminated with blood. Whilst HIV may live for some time outside the body, HIV transmission has not been reported as a result of contact with spillages of blood, semen or other body fluids. Just because someone comes into contact with tiny quantities of HIV in dried blood, it does not follow that infection will occur. Scientists agree that HIV does not survive well in the environment, making the chance of environmental transmission remote. Laboratory experiments have shown that drying, even of very high concentrations of HIV, reduces the amount
Acute infection Chronic Iymphadenopathy

of infectious virus by 90 to 99 percent within a few hours. Therefore, drying of HIV-infected human blood or other body fluids reduces the theoretical risk of environmental transmission to essentially zero. Pathogenesis of HIV Infection The pathogenesis of HIV-1 infection reflects a complex interplay between basal viral load, viral replication, immunodeficiency due to reduction in the T cell counts and the host immune response to the infection. Basal viral load is a primary determinant of rate of the disease progression and prognosis. Numerous studies have confirmed that the factors affecting the infection progression to AIDS and hence the mean survival time include: (a) CD4+ T cell count at the time of infection and (b) Basal viral load. The natural course of HIV infection is shown in Figure 21.6. During the earliest acute phase of the infection, the virus replicates at a very high
Skin and mucous membrane immune defects Systemic immune deficiency

Sub-clinical immune dysfunction

1000 900 800 CD4 T cell concentration 700 600 500 400 300 200 100 0 0 6 12 18 24 30
Viral P24 Antigen

Clinical latency CD8+T Cell

Anti HIV (gp 120) antibodies

CD4+T Cell

36

42

48

54

60

66

72

78

84

Time (months after infection)

Fig. 21.6: Progression of human immunodeficiency virus (HIV) infection

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rate due to the availability of a large number of CD4+ cells. The viral load doubles every 10 hours and peripheral blood viremia reaches above 50,000 copies/ml and may even be as high as 1 107 copies/ml. Rapid multiplication and infection of new CD4+ cells cause a sharp decline in the CD4+ T cells. The symptomatic patients are found to have higher viral load. The viremia peaks at about three weeks of infection and at this time, the p24 (capsid) antigen may also be detected in blood. The virus specific CD8+ cells are also stimulated during this acute phase and might contribute to the decrease in primary viremia. A specific immune response to HIV is triggered primarily in the form of cytotoxic T cell stimulation. This immune attack might check the initial burst of viremia and CD4+ T cells also recover to some extent. The peripheral virus load is reduced significantly; the virus might even disappear from the blood. The infection, of course, continues to spread in the lymph nodes. The virus can be demonstrated to be trapped extracellularly in the follicular dendritic cell network of germinal centers in lymphoid tissues as well as intracellularly, in mononuclear cells. Following this immune attack, the infection becomes Clinically Latent i.e. no signs and symptoms, although virus continues to replicate and CD4+ cell counts in blood continue to decline. Since HIV never stops replicating, there is no true latency of HIV infection. Anti-Gp120 antibodies can be detected during this phase but most of these antibodies are non-neutralizing and hence are ineffective, except in some individuals where neutralizing anti-Gp120 antibodies are found and generally associated with the slow progression of the disease. During this chronic lymphadenopathy phase, the virus load continues to grow, albeit slowly in most patients. It has been estimated that about 10 billion viral particles are produced and one billion CD4+ cells are destroyed everyday during this phase. The continued destruction of CD4+ cells is associated with subclinical immune dysfunction when generalized slow immune response might be observed. Emergence of HIV infection from clinical latency is dependent upon the speed of decline in the CD4+ cell counts and might take up to 15 years to develop, the average being between 8 to 10 years. The decrease in CD4+ cell counts below a critical level (200 cells/ml) is linked with the rise in viremia. By this time, immune system has been heavily compromised and generalized lymphadenopathy could be seen along with the loss of lymph node architecture. This condition was previously described as Persistent Generalized Lymphadenopathy (PGL). Another term used for the preAIDS disease was called as AIDS related complex (ARC). ARC is the symptomatic condition before HIV progresses into overt AIDS and is characterized by fatigue, weight loss, night sweats and superficial fungal infections of the mouth, finger nails and toe-nails. Opportunistic infections and neoplasm are not seen in this condition. The appearance of opportunistic infections marks the progression of the disease into AIDS. About half of the HIV patients also show the syncytia-forming (SI) variants of HIVthese variants being more aggressive towards the CD4+ cells. How Does HIV Evade the Host Immune System? Most of the viral infections are self-limiting and host immune response plays a crucial role in controlling the infections but in case of HIV, the virus continues to proliferate. How does the HIV circumvent the host immune system? The initial infection is very strongly challenged by the cytotoxic T cells. The T-cytotoxic stimulation brings down the viral load in blood. In fact, a minimum (threshold) number of HIV virions must infect to establish sustainable infection otherwise the immune system is able to eliminate it. Once established, HIV (as of today) can not be removed from a person. There are a number of explanations proposed and they might all be contributing in the survival of infection inside the host. Some of them are as follows: a. The first and foremost reason for poor immune response to HIV is its target cells i.e. CD4+

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T lymphocytes. The virus attacks and kills the T helper cells and hence starts weakening the immune system right from the day of infection. The cellular immunity is impaired and slowly gets depleted. b. The virus gets sequestered in the memory T-cells in the lymphoid tissues even when it has been eliminated from the blood. It is very difficult to eradicate the viruses in memory T cells with chemotherapy or immune reaction. c. The HIV is a retrovirus and the reverse transcriptase as well as the RNA polymerase II that catalyze its replication lack any proofreading step and hence give rise to polymorphism. Due to erroneous replication, the virus proteins (including the surface proteins like Gp120 and Gp41) are mutated very fast. Therefore, by the time host immune system prepares itself for an immune reaction against the viral surface proteins, they get mutated. The high frequency of mutations is also responsible for the resistance that HIV shows against the chemotherpeutic reagents. The envelope protein (gp120) can vary as much as 35% in sequence between various HIV groups. In addition, recombinants between different groups are being identified. d. A remarkable feature of the human immunodeficiency virus (HIV) is the dense carbohydrate (glycan) array that surrounds the exposed envelope antigens. HIV genome encodes no gene products capable of synthesizing carbohydrates: its surface antigens are glycosylated entirely by host cellular enzymes. This extensive glycosylation is known to affect almost every aspect of virus biology. The folding of viral glycoproteins, the transmission of the virus and the nature of the immune response to infection. Further, proteins synthesised in the host cell ribosomes undergo extensive glycosylation and myristillization in the Golgi apparatus. The carbohydrates covering on the proteins like Gp120 is Self for the host immune system and hence serve as a Glycan Shield, thus protecting the virus against the host immune system. The coating of HIV with immunologically self glycans (that is, those synthesized by the host cell) is the major mechanism for evading the host immune defense against viral infection. No antibodies against most of the available antigenic surface of HIV are synthesized. e. The trimeric Gp120 molecule is coated by the glycan shield on the outer faces of the protein, the inner immunogenic surfaces are hidden from the immune system due to trimerization along the inner surfaces. f. The GP120 glycosylation sites keep on changing due to errors during its replication, therefore even the glycan shield keeps on changing to evade the immune system. g. Most of the antibodies against HIV are ineffective because, they are directed against non-neutralizing epitopes or exert selection pressure that HIV evades through changes in its envelop sequence. However, some antibodies isolated from infected individuals do show efficacy. An important one is IgG1 2G12 that binds directly and exclusively to gp120 glycans. This antibody binds to the oligomannose cluster on the outer domain of gp120. The epitope is fairly conserved between a broad range of HIVs. Diagnosis of HIV Infection The HIV infection can be detected by a number of diagnostic tests. It is mandatory these days to test all the donated blood for HIV before transfusion. To obtain reliable test results, the test must be performed within four to six hours of taking out the sample from the body in this time duration. Samples kept/stored for more than o 24 to 48 hours, even at 4 C, shall always give erratic and unreliable results as red blood cells start hemolyzing and contamination sets in without fail. In some countries (including India), all patients must be tested for HIV before any kind of surgery. There are several types of tests

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commercially available for HIV testing. The reliable tests are as follows: a. HIV ELISA: The first type and the standard screening test of HIV is ELISA, the HIV antibody test. Antibodies to the HIV envelope and capsid proteins can be detected at about 6 to 12 weeks after the first exposure. The time period between the exposure and the appearance of the antibody test positive in the patient is known as the Window Period. During the window period, people infected with HIV have no antibodies in their blood that can be detected by an HIV test. However, the person may already have high levels of HIV in their blood, sexual fluids or breast milk. Someone can transmit HIV to another person during the window period even though they do not test positive on an antibody test. Most of the modern HIV ELISA kits use a combination of antigens from HIV-1 and HIV2 and hence can detect the presence of either of the two strains with above 99% sensitivity. The major drawback of ELISA tests is their poor specificity. Only 10% of the volunteer blood donors who have a positive ELISA test are confirmed to be HIV patients. ELISA tests can be positive in the absence of disease in infants born to HIV mothers because the baby will carry antibodies from the mother for up to 18 months. False positive ELISA tests could also be due to antibodies against class II antigens, influenza vaccination, acute viral infections and many other conditions and that is why ELISA is repeated at least three times with different kits or different techniques before labeling any sample as positive. b. Antigen testing: The second type of test is an antigen test. The most potent antigen on HIV is the capsid protein P24. Early in the infection, P24 is synthesised in excess and can be detected in the blood serum by a commercial test. As HIV becomes fully established in the body, P24 levels fade to undetectable levels. P24 antigen tests are sometimes used to screen donated blood, but they can also be used for testing for HIV in individuals, as they can detect HIV earlier than standard antibody tests. Some of the most modern HIV tests combine P24 and other antigen tests with standard antibody identification methods to enable earlier and more accurate HIV detection. Antigen testing is particularly useful during the window period. c. Western Blot: The most commonly used confirmatory test for HIV is the Western Blot assay. These assays employ all the three types of viral protein antigens corresponding to each of the three viral genes i.e gag, pol and env. The proteins are separated on the gel by electrophoresis and the bands on the membrane strips are supplied in the kit. The patient sample is tested for antibodies against all of the antigens and is considered positive when it shows the presence of antibodies against all of them. Although Western Blot is an excellent confirmatory test for HIV, it is a poor screening test because it can show the presence of antibodies against one or more HIV antigens even in the absence of HIV infection. Most probable reason for this false positivity is the cross-reactivity. d. Molecular testing: The next type of HIV tests are molecular tests for HIV to check for its DNA or RNA. These types of tests detect the genetic material of HIV itself, and can identify HIV in the blood within a week of infection. DNA/RNA tests come in a number of forms. Babies born to HIV positive mothers may be tested using PCR (Polymerase Chain Reaction). Blood supplies in developed countries are screened for HIV using an RNA test known as NAT (Nucleic Acid-amplification Testing). e. Viral load: When a person already knows that she or he is infected with HIV, they may also have a viral load test to detect HIV genetic material and estimate the level of virus in the blood. DNA/RNA tests are rarely used

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to test for HIV in adults, as they are very expensive and more complicated to administer than a standard antibody or P24 test. Anti-Retroviral (HIV) Therapeutics HIV uses host cell metabolic process for most of its synthetic reactions; therefore it becomes very difficult to interrupt the life-cycle of the virus without killing the host cell. The anti-HIV therapy is designed to target the processes that are unique to the virus so that proliferation of the virus is halted sparing the uninfected cells unharmed. Almost all the stages of the HIV replication cycle are possible targets for chemotherapy e.g. attachment, fusion, reverse transcription, integration, synthesis of envelop proteins, assembly and budding. Azidothymidine (AZT) was the first drug that showed some efficacy against HIV. The drug was actually designed as an anticancer drug (for which it was proved not to be very effective) but soon established itself for its anti-retroviral effects. The compound belongs to the category of nucleoside inhibitors of reverse transcriptase enzyme (discussed below). Following AZT, a streak of new approaches to devise anti-retroviral therapy has been pursued. Most agents are virustatic rather than virucidal. Some of the new approaches are showing promise; however, there are major doubts that eradication can ever be achieved because of the very slow clearance of HIV-infected resting memory T cells. The various types of therapeutic approaches available are discussed below in brief. The single strand HIV RNA genome is first transcribed to double strand DNA by reverse transcriptase, a retroviral-specific enzyme. a. Nucleoside reverse transcriptase inhibitors: These are competitive inhibitors of reverse transcriptase since they bind to the enzymes active site. In addition, they are DNA chain terminators, i.e., they are recognized by reverse transcriptase as nucleotides and incorporated into DNA; however, they terminate the chain elongation as they lack the complete deoxyribose ring or a hydroxyl group at carbon 3 of the sugar. The first of these competitive reverse transcriptase inhibitors was AZT. Being inhibitors of DNA polymerization, they show severe side effects since proliferation of the normal lymphocytes, gut mucosal cells and spermatozoa is also impaired. Some specificity is achieved because AZT has a far higher affinity for reverse transcriptase than it does for the host cell DNA polymerase but it also inhibits cellular enzymes leading to severe side effects. Table 21.4 lists the approved nucleoside RT inhibitors.
Table 21.4: Some of the FDA approved nucleoside RT inhibitors Drug AZT (3'-Azidothymidine) DDI (2',3'-dideoxyinosine) Trade names Zidoudine, Retrovir Didanosine, Videx

DDC (2',3'-dideoxycytidine) Zalcitabine, Hivid d4T (Didehydrothymidine) 3TC (2'-deoxy-3'thiacytidine) Abacavir succinate Tenofovir disoproxil fumarate Stavudine , Zerit Lamivudine, Epivir Ziagen Viread

b. Non-nucleoside reverse transcriptase inhibitors: The non-nucleoside reverse transcriptase inhibitors are non-competitive allosteric inhibitors of the enzyme, i.e. they bind the enzyme at a site other than the active site. These are the most potent and selective reverse transcriptase inhibitors that work at nanomolar concentrations. Unlike nucleoside analogs, they have minimal toxicity in tests with cultured cells, although they show some toxicity when used in humans. They work synergistically with nucleoside analogs such as AZT. There is now a collection of such agents that are chemically distinct. The three drugs of this type approved in the U.S. are nevirapine, delavirdine and efavirenz.

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c. Protease inhibitors: Protease is the enzyme that cleaves the viral polyproteins into the functional proteins before they are assembled into new virions. Many protease inhibitors are being developed but at present, only a few are approved. They are all substrate analogs i.e. they mimic a peptide that can bind to the active site of the viral protease. When used individually, protease inhibitors can drive down viral load to between one 30th and one 100th of initial value. Some of the approved protease inhibitors are listed in Table 21.5.
Table 21.5: FDA approved protease inhibitors Drug Saquinavir Saquinavir mesylate, SQV Ritonavir, ABT-538 Lopinavir and Ritonavir Indinavir, IDV, MK-639 Nelfinavir mesylate, NFV Amprenavir Fosamprenavir calcium Tipranavir Atazanavir sulfate Brand Name Fortonase Invirase Norvir Kaletra Crixivan Viracept Agenerase Lexiva Aptivus Reyataz

The above three major categories of antiHIV drugs are effective against HIV but result in development of resistant strains within a few weeks to months. Therefore, combination therapy is used which is found to be reasonably successful in controlling the disease. The combinations used are known as Highly Active Anti-Retroviral Therapy (HAART). d. Other anti-HIV drugs: Apart from HAART, the drug groups which are under various stages of development are: i. Drugs that inhibit uptake of the virus: The uptake of the virus involves CD4 antigen, the receptor, and chemokine

receptor co-receptors. Blocking the interaction of HIV gp120 with either of these could potentially inhibit infection. Agents that bind to either receptor or gp120 might be useful. CD4-Ig2 (PRO542) PRO 542 is a tetrameric form of soluble CD4 antigen genetically fused to an immunoglobulin. This CD4-immunoglobulin fusion protein comprises the D1 and D2 domains of human CD4 and the heavy and light chain constant regions of human IgG2. It has a high affinity for gp120. CD4-PEG (Pseudomonas aeruginosa endotoxin A) CD4-PEG drug is synergized with AZT but may have non-specific toxicity. ii. Antibodies that bind to CD4 antigen: Antibodies that bind to CD4 antigen are likely to block virus attachment but may be immunosuppressive because they will lead to depletion of CD4 cells. iii. Agents that block interaction of gp120 with co-receptors: CCR5 antagonists: CCR5 is a co-receptor for HIV and CCR5 deletions make the infected patients resistant to HIV. Persons with this deletion appear normal and healthy. This suggested that targeting CCR5 might block HIV in patients without any severe effect on the immune system. RANTES is a chemokine that binds to CCR5 and a RANTES analog (n-nonanoyl (NNY)RANTES) has been shown to block HIV infection. Although effective, RANTES also has resistance problems as its use results in development of virus strains which are resistant to the RANTES analog and can use the CXCR4 coreceptor. Moreover, chemokine derivatives are proteins (or at least peptides) and so will not be orally available. Small molecule co-receptor antagonists have been found. These are often highly negatively charged molecules. AMD3100 is undergoing clinical trials. It

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appears to bind to CXCR4 (fusin) and block the interaction between CXCR4 and the V3 loop of Gp120. iv. Agents that block fusion by interacting with gp41: Fuzeon: Peptides derived from gp41 can inhibit infection, probably by blocking the interaction of gp41 with cell membrane proteins during fusion or by stopping the conformational change that results from the association of two gp41 molecules and which is necessary for fusion. T-20 (Fuzeon) blocks this conformational change. In clinical trials, a nearly two log reduction in plasma viral levels was achieved. Peptides containing D-amino acids that would fit a cavity on the Gp120 molecules have been identified which do inhibit fusion. v. Drugs that inhibit integrase activity: Integration of the viral DNA into the host cell chromosome is essential for the production of new virus particles and this would be an excellent target for a specific anti-viral agent since there is no homologous enzyme in humans. A new class of anti-retroviral drugs targets the integration enzyme, the integrase. The examples include Zintevir (AR177) and Lchicoric acid. vi. Drugs that block post-translational modifications: The matrix protein is myristoylated and the surface glycoprotein is glycosylated. These posttranslational modifications offer a potential target for drug therapy. Blockers of myristoylation: Analogs of myristic acid that block myristoyl CoA:Myristoyl transferase such as 4oxatetradecanoic acid are non-toxic and work well in cultures. Clinical efficacy is unknown. Blockers of glycosylation: Castanospermine, a naturally occurring alkaloid and inhibitor of glucosidase-I has some effect against HIV in vitro. This has led to the development of analogs such as N-butyl deoxynojirimycin (butyl-DNJ) and 6-butyl-castanospermine which have more potent activity against HIV. vii. Nucleic acid based strategies: Recently, another approach to chemotherapy has been discovered i.e. using nucleic acids that selectively bind or neutralize the viral genes. Several possibilities are being investigated. These include: Anti-sense RNAs that bind to HIV mRNAs and block their translation. Catalytic RNAs (ribozymes). Oligonucleotides that target the nucleic acid-binding control proteins of HIV such as TAT and REV. Approaches to Vaccine Development Since the development of an effective vaccine against smallpox, many diseases caused by viruses are no longer a major public health problem or their incidence has been greatly reduced. Initially, when the causative agent for AIDS was discovered to be a virus, it was hoped that a protective vaccine would soon be available. However, the AIDS epidemic has been with us for more than twenty years and we still have no effective vaccine, despite almost 100 clinical trials. The Problems Involved in the Production of an Anti-HIV Vaccine Apart from the points discussed above regarding evasion of the immune response by HIV, the following road-blocks have to be circumvented to develop an effective vaccine against HIV. A large number of government as well as private projects are underway to achieve this objective. 1. HIV is a retrovirus. A vaccine strain that protects against AIDS will still have LTRs and may still be oncogenic. 2. HIV is a retrovirus...The use of reverse transcriptase and RNA polymerase II which have no proof reading activity leads to rapid

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polymorphism. Therefore, a vaccine against a laboratory strain will be unlikely to affect the myriad strains in the population. Antibodies against HIV elicited by a vaccine may increase uptake by macrophages. Antibodies against HIV elicited by vaccine may give rise to autoimmunity. We still do not know completely how HIV leads to CD4+ T4 cell depletion. W e have no good animal models. Chimpanzees can be infected but do not develop AIDS, so early trials on human beings become necessary in vaccine development. Alternatively, for development of a vaccine, we can use SIV and monkeys but this is not the same virus. Cell to cell transmission may make standard humoral antibodies elicited by a vaccine unimportant. antibodies but this also means that the large number of oligosaccharide chains and the folding of the protein itself shields potential epitopes. One site that presumably cannot change too much as a result of mutation is the receptor binding site of gp120 (i.e. either the CD4 antigen binding site or the coreceptor binding site) since mutation would preclude virus-cell interaction. Nevertheless, these sites remain buried in the gp120 molecule. One interesting approach has been to design vaccines against epitopes that are exposed only after the conformational change that occurs when CD4 antigen and gp120 interact. So, the glycoprotein-based subunit vaccines have not been successful and are less likely to succeed. Nevertheless, it is apparent that neutralizing antibodies against HIVgp120 or gp41 can be raised. b. Cell-mediated immunity: It is known that the major drop in HIV virions soon after infection is the result of a cytotoxic T cell response rather than a humoral response. Thus, attempts are being made to display HIV epitopes on cells in association with HLA molecules and destroy infected cells rather than free virions. c. Whole virus (killed) vaccines in animals: In early studies with the SIV/Macaque and Chimpanzee models, a whole virus vaccine was produced. Initially, there appeared to be some protection but it became clear that this kind of vaccine gives protection against the same virus that is used for making the vaccine. A successful vaccine will need to act against the myriad substrains of HIV that arise during a normal infection or will need to neutralize the initial infecting virions. Using a killed virus preparation, a very complex vaccine that would elicit antibodies against numerous strains of the virus would be required. The early whole virus vaccine studies were done with syncytium-inducing virus (which

3. 4. 5. 6.

7.

Strategies for an HIV Vaccine a. Subunit vaccines: Vaccines using purified proteins do not induce a cell-mediated response; rather, they stimulate neutralizing antibodies which may lead to sterilizing immunity. Most current anti-HIV vaccines fall into this category. They consist of recombinant proteins (e.g. gp160 or gp120) presented in a soluble form. Original attempts at making a vaccine in chimpanzees used soluble gp120 antigen and it was found that immunized chimps can resist subsequent intravenous challenge by virus of the same type from which the antigen was made. However, as has been noted, HIV is constantly changing and challenge by other types showed that there was no protection against these. The immune response generated by vaccines that stimulate neutralizing antibodies is usually short-lived (often a matter of weeks) and of low titer. Among the reasons for the poor response is the necessity for the gp120 to remain in its natural conformation, particularly its trimeric state, to elicit neutralizing

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binds to X4 co-receptor) but non-syncytiuminducing viruses are the infectious form. Clearly, we require a vaccine that acts against the initial infection by non-syncytium inducing virus (that bind to the R5 coreceptor). Attempts have been made to do this but primary HIV isolates are a problem because they seem to be far better at concealing their neutralization sites from antibodies by burying vulnerable epitopes within the protein. The epitopes are exposed during fusion of the virions with CD4 cells, therefore, fusion molecules were isolated from laboratory cell lines carrying the receptor and used to make antibodies in mice. The antibodies that the mice produced neutralized 23 of the 24 primary isolates, many of which were non-syncytium-inducing. d. Attenuated virus vaccines: There have been several demonstrations that an attenuated whole virus vaccine can protect monkeys against subsequent challenge by SIV. There are natural attenuated forms of HIV that might be good candidates for vaccine development. Some people, who are actively infected and show no signs of AIDS, harbor HIV NEF deletion mutants. For example, there is a cohort of Australians who have an HIV strain with multiple deletions in the NEF/LTR region of the genome. All of these people got the virus from transfusions and had shown no symptoms after more than 15 years. This led to the idea that an attenuated HIV with an NEF deletion would be a good potential vaccine. It was found that NEF deletion mutants of SIV can very effectively protect monkeys against simian AIDS without causing the disease. However, a natural NEF/ LTR deletion mutant was found to be pathogenic for humans who were exposed to it via a blood transfusion. It resulted in immunosuppression although onset was later than with the wild type virus. It also appears that there is still the problem of reversion, even if it is small, and unfortunately this has occurred. A NEF deletion of SIV reverted to a virulent strain by the process of gene duplication of an adjacent sequence. Moreover, juvenile monkeys developed AIDS when given a high dose of the vaccine. So there might be good chance of an infection among vaccine recipients, especially those who are immune compromised (cancer patients or the elderly). It is thought that the vaccine strain may continue to replicate at low levels in some compartment of the immune system. e. Recombinant vaccines: In order to present antigen in the context of HLA molecules and raise a cell-mediated response, recombinant vaccines that contain a gene (or genes) from HIV in a non-pathogenic vector have been developed. One vector that has been tried is vaccinia, the live virus that is used as a smallpox vaccine. The results have been disappointing. The anti-HIV vaccine based on the Ankara vaccinia strain elicits both humoral and cellmediated immunity and seems effective in animals. Unfortunately, in humans there was only limited anti-HIV activity, perhaps because most of us have immunity to vaccinia, a result of smallpox vaccination. Another vector, canarypox, has been used as the basis of an HIV recombinant vaccine. Again, those tested have shown limited development of a long-lived cell-mediated response. In one trial (2007), a canarypox vaccine was tested along with administration of interleukin-2 to boost the patients immune response. The subjects were HIV positive and being treated with HAART. The ALVAC vCP1452 vaccine contains several HIV-1 genes: gp120, expressed by a part of the env gene of the MN HIV-1 strain; the anchoring transmembrane domain of gp41 of the LAI HIV-1 strain; the p55 polyprotein expressed by the gag gene of the LAI HIV1 strain; part of the pol gene expressing

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protease activity from the LAI HIV-1 strain in order to process the p55gag polyprotein; in addition there are genes expressing peptides from pol and nef that are HLA-A2-restricted cytotoxic T-cell lymphocyte epitopes. Unfortunately, there was no difference between the treatment and the placebo group. Another possible vector is replicationdefective adenovirus (pseudovirions). These viruses can infect cells and are expressed but progeny virions are not formed. Some vaccines contain multiple viruses, each expressing different HIV proteins such as nef, gag and pol. These offer protection in animal models but there is always the question of how relevant such models are to human trials. HIV Vaccines: Current Status Live attenuated vaccines based on deletion of non-essential NEF gene in monkeys show that a vaccine can be developed against SIV. The vaccine is effective against highly pathogenic strains of SIV. Because of the problems with reversion, the deletion strategy involved making vaccine strains with multiple NEF deletions. These vaccine strains were found to cause disease in juvenile monkeys; adult monkeys with the vaccine strain also subsequently showed symptoms of disease. Whole killed virus vaccine is again being studied (see above). The problem remains, however, that failure in inactivation would cause serious disease. Also, this is a retrovirus that has the potential to integrate into the host genome and cause disease at a much later time. Recombinant subunits of the envelope and core do induce high levels of neutralizing antibody but there is poor cross-neutralization and there are no killer T cells to attack infected cells. Peptide epitopes directed against the principal neutralizing domain of HIV gp120 have been tried but elicit no cell-mediated immunity. DNA immunization is safe, inexpensive and transportable. It should give both humoral and cell-mediated immunity. The HIV DNA, using antigens from env and core region of the virus, can be incorporated into a harmless plasmid. The DNA vaccines have already been effective against SIV and are being tested in humans. At this time you should know the following: 1. Pandemic of human immunodeficiency virus. 2. Properties of lentiviruses and molecular structure of HIV. 3. Life-cycle and pathogenesis of the HIV. 4. Testing and therapeutic approaches to control HIV infection. 5. Latest in the development of a vaccine against HIV.

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