Human immunodeficiency virus: An overview

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The History of HIV Discovery


Human immunodeficiency virus (HIV), supposed to be derived from primate lentiviruses, is the etiological agent of HIV infection with acquired immunodeficiency syndrome, or AIDS.

The infection was first registered in 1981, and its causative retroviral agent was primarily isolated by the end of 1983 by the French virologists F. Barre-Sinoussi and L. Montagnier. To confirm the results of discovery, this viral isolate was sent to a highly experienced US laboratory headed by R. Hallo that specialized in retrovirus study. At the end of 1983 the research group of F. Wong-Staal working under the guidance of R. Hallo reported about isolation of a new retrovirus – putative causative agent of AIDS. Further molecular genetic analysis demonstrated the identity of genomes of both viral agents.

In 2008 F. Barre-Sinoussi and L. Montagnier were awarded Nobel Prize in Physiology or Medicine for their pivotal discovery. In 1986 in West Africa F. Clavel and coworkers discovered another species of human immunodeficiency virus – HIV-2.


Classification of Retroviruses


Retroviridae family of RNA-containing viruses embraces 2 subfamilies (Orthoretrovirinae and Spumaretrovirinae) with 7 viral genera. All the representatives of family Retroviridae contain unique reverse transcriptase enzyme, which synthesizes DNA copy of viral genome on the base of viral RNA template.

HIV viruses (species HIV-1 and HIV-2) belong to subfamily Orthoretrovirinae and Lentivirus genus, which encompasses the viruses, capable of causing long-lasting slow viral infections. Lentiviruses have been isolated from many living species, including 20 different primate species.

As some other RNA-containing viruses, HIV demonstrates outstanding genetic variability. HIV-1 is divided into genetic groups (M, N, O, and P). More than 90% of cases of HIV-infection are caused by representatives of M (main) viral group.

Every genetic group contains multiple genetic subtypes. For instance, M group includes above 10 subtypes (A-K). Subtypes are capable of cross-recombination with the formation of circulating recombinant forms of HIV. HIV-2 has 9 genetic groups (A-I). Subtype variations generate multiple HIV quasispecies during the course of HIV infection.


Structure of HIV


HIV viruses are spherical in shape, 80-100 nm in diameter with cylindrical cone-shaped core. Virion is covered with lipid envelope, carrying receptor glycoproteins. Under lipid coat protein shell of p17 protein is present.

Viral genome is composed of single stranded positive-sense RNA. The genome is diploid that means the presence of two equal (+) RNA molecules. HIV contains three genes encoding viral structural proteins – gag, pol, and env.

Viral env gene encodes major viral envelope proteins. Glycoprotein gpl20, product of env gene, contains binding domains for virus attachment to host CD4 molecules and coreceptors, and carries the main antigenic determinants that elicit neutralizing antibodies.

Glycoprotein gp4l of env gene contains both a transmembrane domain that anchors the glycoprotein gp120 in the viral envelope and a fusion domain that facilitates virus penetration into target cells. Combination of gp41 and gp120 results in complex gp160 glycoprotein, integrated in the viral envelope.

Viral pol gene codes for viral enzymes reverse transcriptase (p66), protease (p32), and integrase (p11). Gene gag (group specific antigen) encodes primary polyprotein with inner capsid peptides (e.g., p17 and p24). After translation viral polyprotein is cleaved by viral protease resulting in structural proteins.

Six additional genes encode proteins that regulate viral reproduction in vivo participating in pathogenesis of HIV infection:

– Tat protein (product of tat gene) activates reverse transcription of viral RNA;

– Rev protein (product of rev gene) regulates transcription of viral structural proteins;

– Nef protein (product of nef gene) inhibits expression of CD and HLA molecules on membranes of infected cells; elicits chemokine production by macrophages that activate resting T cells resulting in productive HIV infections;

– Vif protein (product of vif gene) suppresses synthesis of antiviral proteins;

– Vpr protein (product of vpr gene) stimulates replication of viral RNA and transcription of viral protens;

– Vpu protein (product of vpu gene) promotes virion release;

– Vpx protein of HIV-2 (product of vpx gene) stimulates reverse transcription of HIV-2 RNA.


Virion Resistance


Despite generally low environmental resistance of retroviruses, in certain conditions and in high concentrations HIV maintains viability for a long time (e.g., above 10 days in blood in syringes at room temperature or at 4оС for 2-4 weeks). When dried in blood spots, HIV stays viable for several days.

HIV remains infectious during long-term storage at low temperatures (-20оС and less). Virus is inactivated by heating at 60°C for 30 min and at 100оС for 1 min.

HIV is sensitive to the most common disinfectants and antiseptics (household bleach, sodium hypochlorite and other halides, hydrogen peroxide, aldehydes, ethanol, detergents, and others). The virus is also killed by acidic or alkaline pH.


Viral Replication Cycle


Virus attachment is performed via gp120 receptor that binds to CD4 molecules of lymphocytes, macrophages, and some other cell types.

The highest density of membrane CD4 is essential for CD4+ T helper cells and T memory cells, but expression of CD4 on monocytes, macrophages, dendritic cells, glial cells, astrocytes and neurons of CNS, or enterocytes is enough for HIV adherence.

A second co-receptor in addition to CD4 is necessary for HIV-1 to gain entry to the cells. It is required for fusion of the virus with the cell membrane. Virus first binds to CD4 and then to the coreceptor. These interactions cause conformational changes in the viral envelope that induces gp41-mediated membrane fusion and virus uptake.

Host chemokine receptors serve as HIV co-receptors – CCR5 is the predominant co-receptor for macrophage-tropic strains of HIV, whereas CXCR4 is the co-receptor for lymphocyte-tropic strains of HIV.

Uncoating of HIV occurs in cytoplasm. Here viral genomic RNA undergoes reverse transcription to complementary DNA by enzyme reverse transcriptase.

Viral DNA is imported to the nucleus for integration to the host genome (provirus state). Incorporation of HIV DNA is catalyzed by viral integrase.

HIV replication largely occurs in activated T cells and to a lesser extent in macrophages. They are stimulated by TNF-alpha and other proinflammatory cytokines followed by activation of cellular transcription factors (mainly, NF-κB) that trigger the transcription of viral RNA.

After ribosomal translation primary viral polyproteins are processed by viral protease. Viral assembly (morphogenesis) takes place in the cytoplasm; progeny virions leave the cell by budding. Complete duration of HIV replication cycle averages 2.5 days.

As it was mentioned above, HIV demonstrates highest genetic variability. Because of rapid viral proliferation and the error-prone mode of activity of HIV reverse transcriptase, every nucleotide of the HIV genome probably undergoes daily mutation.


Epidemiology, Pathogenesis, Clinical Findings and Immunity in HIV Infection


From its first case, HIV infection and AIDS has become a global epidemic, affecting different populations and all geographic regions. Once infected, individuals remain infected lifelong. Within a decade, if left untreated, the vast majority of HIV-infected persons develop fatal opportunistic infections resulting from HIV-induced suppression of the immune system. The source of infection is a HIV-infected person.

The most common route of transmission is sexual intercourse (homo- and heterosexual contacts) that accounts for more than 70% of infection spread. Next follows parenteral transmission (artificial route) that predominantly includes intravenous drug use. Occasionally the infection is contracted after medical manipulations, predominantly, blood transfusions.

Mother-to-child or vertical transmission occurs during pregnancy or childbirth, and through breastfeeding with maternal milk. Transmission of HIV-2 is 10-20 times less frequent than HIV-1 in the same conditions of exposure to virus. HIV infection affects CD4-bearing cell populations.

The main target of virus action is the subset of T helper lymphocytes, which express CD4 phenotypic marker on their surface. It was determined that HIV co-receptor CXCR4 is carried by “naive” T cells, whereas another co-receptor CCR5 is expressed predominantly by macrophages and dendritic cells as well as by activated T lymphocytes and memory T cells.

After viral entry reverse transcription of viral genomic RNA leads to complementary DNA synthesis and its further integration with cell genome resulting in provirus state. Proviruses remain long-time associated with affected cells, but under a number of external and internal stimuli viral replication is activated. Various cytokines (e.g., TNF-alpha or IL-10) induce HIV transcription in both macrophages and T cells.

Chronic activation of the TNF-signaling pathway enhances HIV-1 transcription. Viral propagation within T helpers causes cell death due to multiple devastating effects of virus:

– cytopathic effect on T cells with syncytium formation;

– direct and TNF-mediated apoptosis of T cells;

– pyroptosis, or inflammatory T cell death via activation of caspase-1;

– death of infected T cells under the attack of cytotoxic T cells and NK cells;

– general inhibition of hematopoiesis.

Monocytes and macrophages play a substantial role in the dissemination of HIV infection. Unlike CD4+ T lymphocytes, monocytes are relatively refractory to the cytopathic effects of HIV, and the virus can be transported to various organs and tissues (such as the lungs and brain).

Infection of glial cells, astrocytes and neurons of CNS is followed by permanent neuropsychiatric disorders in patients with HIV infection. Progressive decline of T helpers leads to profound attrition of the immune system that affects basic functions of cytotoxic T cells, natural killer cells, impairs secretion of cytokines, etc.

As the final result, a tremendous variety of opportunistic infections arises in HIV-infected person due to deep immunosuppression, caused by HIV. A typical course of untreated HIV infection spans about a decade. Stages of HIV infection include:

incubation period;

acute infection;

clinical latency;

persistent generalized lymphadenopathy;

AIDS development;

death of patient resulted from AIDS-associated diseases.

During incubation in 24 h after exposure HIV invades dendritic cells at the portal of entry; in 24-48 h infected dendritic cells migrate into regional lymph nodes; in 4-11 days HIV appears in blood resulting in viremia; and in 3-4 weeks first clinical signs of infection arise.

Acute infection is characterized with dissemination of virus to lymphoid organs. An acute mononucleosis-like syndrome develops in many patients (50-75%) 3-6 weeks after primary infection. There is a significant drop of circulating CD4 T cells at early time. An immune response to HIV occurs 1 week to 3 months after infection, plasma viremia declines, and the number of CD4 cells restores. However, the immune response is unable to clear the infection completely, and HIV-infected cells persist within the lymph nodes.

The period of clinical latency may last for as long as 10 years. During this time, there is a high level of ongoing viral replication. Coming next persistent generalized lymphadenopathy is characterized with gradual enlargement of different groups of lymph nodes.

And eventually, the patient will develop clinically manifested disease with opportunistic infections or neoplasms. They emerge due to severe immune system failure resulting in acquired immunodeficiency syndrome, or AIDS. The predominant sources of morbidity and lethality among AIDS patients are opportunistic infections, induced by pathogenic agents that rarely cause serious diseases in immune-competent individuals.

The most common opportunistic infections in AIDS patients are caused by:

bacterial pathogens – Mycobacterium tuberculosis, Mycobacterium avium-intracellulare, Listeria monocytogenes, salmonella species, streptococcus species and many others;

viruses – cytomegalovirus (CMV), varicella-zoster virus, herpes simplex viruses, adenoviruses, etc.;

protozoans – Toxoplasma gondii, Cryptosporidium spp.;

fungi – Candida albicans, Cryptococcus neoformans, Histoplasma capsulatum, and Pneumocystis jirovecii.

Tuberculosis is the major AIDS-associated disease in HIV-infected persons. It develops at least in 30-40% of HIV-infected individuals being the major cause of patient’s death (25-40% of total AIDS lethality). Global spread of HIV and tuberculosis co-infection is sometimes called as “Syndemic”.

Severe AIDS-indicator diseases are also associated with mycobacterioses, systemic candidiasis, CMV- and varicella-zoster viral infections, pneumocystis pneumonia and T. gondii infection.

AIDS patients exhibit a marked predisposition to the development of cancer. AIDS-associated tumors include lymphomas, Kaposi’s sarcoma, (vascular neoplasm, caused by herpesvirus type 8, thought to be of endothelial origin), cervical cancer, etc.

Neurological abnormalities are common in AIDS affecting 40-90% of patients. They are manifested by HIV encephalopathy, peripheral neuropathies, and most serious, AIDS dementia complex.

HIV-infected persons develop both humoral and cell-mediated immunity against the virus. Antibodies to a number of viral antigens (p24, gp41, gpl20) develop soon after infection, but they can’t stop infection progressing. Cellular responses (cytotoxic T cells and natural killers) can destroy virus-infected cells.

Nevertheless, immune response to HIV isn’t able to eliminate virus. AIDS consequences, predominantly opportunistic infections and CNS disorders, lead to inevitable death of AIDS patient.

The evident devastating nature of HIV infection multiplied by its rapid spread has posed a tremendous threat to global health security. In 2005 Joint United Nations Programme on HIV/AIDS (UNAIDS) in its annual special report characterized HIV infection as “…one of the most destructive epidemics in recorded history”.

On the peak of AIDS pandemic (1996-2005) a total number of HIV-infected persons became closer to 40 million, about 2-3 million people annually died from AIDS-associated diseases, and the annual number of individuals newly infected with HIV exceeded 4 million.

According to WHO data, more than 34 million people died from the start of AIDS pandemic. In September 2000 Member States of United Nations at the Millennium Summit adopted United Nations Millennium Declaration. It included 8 crucial international development aims – the Millennium Development Goals (MDG).

The Goal 6 claimed “…to halt and begin to reverse the spread of HIV/AIDS by 2015”. It was appended in 2006 with a new AIDS target “…to achieve universal access to antiretroviral medicines for people in need by 2010”.

As the result, only owing to outstanding joint efforts of national governments and global international organizations working under the aegis of UNAIDS (WHO, UNESCO, UNDP, UNICEF, World Bank and others) it has become possible to break down the progression of HIV pandemic.

This great success was largely related with expanding availability of highly efficient antiretroviral therapy for HIV-infected people.

In July 2015 the report of UNAIDS entitled “How AIDS changed everything” with preface of UN General Secretary Ban Ki-moon stated: “The world has achieved the AIDS targets of Millennium Development Goal 6. The epidemic has been halted and reversed”.

It has been confirmed by the next data – the number of persons newly infected with HIV globally declined by 35% in 2000–2014; the number of people dying from HIV-related diseases declined by 41% since 2004, the peak epidemic year; and almost 16 million people acquired HIV treatment.

The current number of HIV-infected individuals is equal now to 36.9 million.

UNAIDS report has also emphasized a future challenging objective as essential part of the sustainable development goal – ending the AIDS epidemic by 2030.

Despite undoubted achievements in the combat with HIV infection, a lot of issues haven’t been solved yet.

First of all, the total number of HIV-infected persons still remains great. Most of the suffering people (estimated 25.8 million) live in Sub-Saharan Africa, the region “most heavily affected by the epidemic”. An estimated 5 million HIV-infected people live in Asia and the Pacific region.

Second, antiretroviral treatment should cover at least 60-80% of all HIV-infected persons to maintain positive tendency of global HIV decline.

And finally, certain world regions demonstrate increasing trends in HIV spread; among them are the countries of Eastern Europe and Central Asia, including Russian Federation, Ukraine, and Belarus.


Laboratory Diagnosis of HIV Infection


Diagnosis of HIV infection is established only on the base of highly specific and sensitive laboratory tests that detect specific antiviral antibodies or viral nucleic acid in clinical specimens (primarily, patient’s blood or serum).

Serological testing is the most suitable in clinical practice to determine HIV infection. Antibodies to HIV are evaluated by enzyme-linked immunosorbent assay (ELISA). If properly performed, these tests have a sensitivity and specificity exceeding 98%.

The first positive ELISA test of a serum sample must be confirmed by a repeat testing.

If the repeat ELISA is reactive, a confirmatory test is performed. The most widely used confirmation assay is the western blot technique, in which antibodies to various HIV proteins of specific molecular weights should be simultaneously detected. Antibodies to viral core protein p24 or envelope glycoproteins gp41, gpl20, or gpl60 are most commonly assessed.

The majority of individuals display seroconversion (reveal antibodies) within 2 months after viral exposure. HIV infection for longer than 6 months without a detectable antibody response is very rare.

Current modifications of ELISA test for diagnosis of HIV infection allow simultaneous determination of viral antigens and antiviral antibodies in the same clinical specimen.

Simple and rapid tests for detecting HIV antibodies are also based on latex-agglutination.

Amplification assays such as the real-time reverse transcriptase PCR and hybridization tests are also commonly used to detect viral RNA in clinical specimens. HIV RNA level (viral load) is an important predictive marker of disease progression and valuable tool to monitor the efficacy of antiviral therapy.

Other auxiliary laboratory tests for AIDS diagnosis include various methods of assessment of immune status. CD4+ T helper subset count is evaluated by flow cytometry or immunofluorescence with monoclonal anti-CD4 antibodies.

CD4+ cell count (normal level – 800-1000 cells/μL of blood) shows direct correlation with AIDS progression – falling below 500 cells/μL predisposes to development of opportunistic infections; the decline less than 200 cells/μL corresponds to AIDS.

Diagnosis of AIDS-associated opportunistic infections rests on rapid molecular tests (e.g., PCR) followed by pathogen isolation and identification.


Principles of HIV Treatment and Prophylaxis


Practical implementation of HIV chemotherapy with combinations of various antiretroviral drugs, referred to as highly active antiretroviral therapy (HAART), literally revolutionized the treatment of HIV-infected persons.

HAART suppresses viral replication below the limits of detection in plasma and decreases the viral load in lymphoid tissues that leads to the recovery of immune response.

However, HAART has failed to cure HIV infection. DNA copy of viral genome is harbored within long-living infected cells, including memory CD4+ T cells and probably macrophages and seminal cells. When HAART is discontinued or there is a treatment failure, virus production reactivates. Thus, the treatment should last lifelong.

Nonetheless, if HIV patients strictly follow treatment regimens, their life expectancy comes closer to average lifespan of human population.

Antiretroviral drugs with various mechanisms of action are included into HAART treatment scheme:

nucleoside inhibitors of HIV reverse transcriptase (lamivudine, abacavir, azidothymidine and many others);

non-nucleoside inhibitors of HIV reverse transcriptase (efavirenz, nevirapine);

– HIV protease inhibitors (indinavir, nelfinavir, ritonavir);

– HIV integrase inhibitors (raltegravir, elvitegravir);

inhibitors of HIV entry and fusion:

maraviroc – inhibits binding of gp120 to human CCR5 co-receptor;

enfuvirtide – binds to HIV gp41 receptor, thereby blocking viral fusion.

Initial HAART schedule presumes administration of 2 nucleoside inhibitors of HIV reverse transcriptase and one drug from other antiretroviral groups (e.g., HIV protease inhibitor).

Whereas monotherapy usually results in the rapid emergence of drug-resistant mutants of HIV, combination therapy, which targets multiple steps in virus replication, prevents the emergence of HIV quasispecies.

Post-exposure prophylaxis of HIV infection (PEP) is administered after occasional contact with biological fluids of HIV-infected persons (unprotected sexual intercourse, accidental needlestick injury, scalpel cut, infected blood transfusion, etc.). To be maximum effective, PEP must be initiated without any delay (within first 72 hours after the exposure). Modern schemes based on HAART are currently used including HIV integrase inhibitors. After primary PEP it is recommended to continue antiretroviral therapy at least for 4 weeks. The repeat laboratory testing of affected persons is performed up to 6 months after exposure.

Specific prophylaxis of HIV infection is not created yet. A safe and effective vaccine is the best tool of controlling the AIDS pandemic. Recombinant viral envelope glycoproteins are the most likely candidates for vaccine.

Unfortunately, vaccine development is extremely difficult because HIV mutates rapidly; furthermore, the virus stays integrated within genome of many infected cells and thereby not completely eliminated by host immune response.

Non-specific prophylaxis presumes the maintainance of a lifestyle that minimizes or eliminates the high-risk factors of HIV spread. This is directly related with the success of educational projects offering behavioral changes.